WO2019141265A1 - 用于靶向活化cd44分子的胶束纳米载体递送系统、其制备方法和用途 - Google Patents

用于靶向活化cd44分子的胶束纳米载体递送系统、其制备方法和用途 Download PDF

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WO2019141265A1
WO2019141265A1 PCT/CN2019/072471 CN2019072471W WO2019141265A1 WO 2019141265 A1 WO2019141265 A1 WO 2019141265A1 CN 2019072471 W CN2019072471 W CN 2019072471W WO 2019141265 A1 WO2019141265 A1 WO 2019141265A1
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vulnerable plaque
peg
delivery system
plaque
nanocarrier
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PCT/CN2019/072471
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French (fr)
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马茜
孙洁芳
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北京茵诺医药科技有限公司
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Priority to CN201980001820.3A priority Critical patent/CN110545794B/zh
Publication of WO2019141265A1 publication Critical patent/WO2019141265A1/zh

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Definitions

  • the invention belongs to the technical field of targeted drug delivery, and in particular relates to a nanocarrier for targeting activated CD44 molecules, in particular to vulnerable fragile plaques, especially a micelle (Micelle) nano delivery system.
  • the invention also relates to the preparation and use of the nanocarriers, in particular the micelle delivery system, in particular in the diagnosis, prevention and treatment of vulnerable plaques or diseases associated with vulnerable plaques.
  • Vulnerable plaque refers to atherosclerotic plaques that have a tendency to thrombosis or are likely to progress rapidly into "criminal plaques", including Rupture plaques, aggressive plaques, and partially calcified nodular lesions.
  • the techniques currently used for the diagnosis of vulnerable plaque mainly include coronary angiography, intravascular ultrasound (IVUS), and laser coherence tomography (OCT), but these techniques are all invasive, and the diagnostic resolution and The accuracy is not high, and these diagnostic techniques are expensive, which also limits the clinical popularity to a certain extent. Therefore, there is an urgent need for non-invasive diagnostic techniques and formulations for vulnerable plaque.
  • the current treatment of vulnerable plaques is mainly systemic administration, such as oral statins (hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors), aspirin, matrix metalloproteinases (MMPs) inhibition Agents and/or fibrates, etc.
  • HMG-CoA hydroxymethylglutaryl coenzyme A
  • MMPs matrix metalloproteinases
  • statins commonly used in clinical practice are relatively low, such as ⁇ 5% for simvastatin, about 12% for atorvastatin, and about 20% for rosuvastatin.
  • Animal experiments have also confirmed that when the dose of statin is increased to more than 1 mg/kg, it can increase the thickness of the fibrous cap and reduce the volume of plaque, which makes the stability and reversal of oral administration of statins. The effect of the block has encountered a bottleneck.
  • a targeted drug delivery system refers to a drug delivery system that has the ability to target administration. After administration via a route, the drug contained in the targeted drug delivery system is specifically enriched in the target site by a vector with a targeting probe.
  • the targeted drug delivery system is capable of targeting the drug to a particular lesion site and releasing the active ingredient at the target lesion site. Therefore, the targeted drug delivery system can make the drug form a relatively high concentration in the target lesion site, and reduce the dose in the blood circulation, thereby improving the drug effect while suppressing toxic side effects and reducing damage to normal tissues and cells.
  • nanocarriers commonly used in targeted drug delivery systems are liposomes.
  • liposomes have the advantage of improving the drug efficacy and reducing the toxic side effects of the drug, due to poor stability in vivo, the cycle time is insufficient, and finally the bioavailability of the drug is limited.
  • the in vitro stability of the liposome is also insufficient, and the phospholipid is easily oxidatively hydrolyzed during storage, and the liposome vesicles are easily aggregated and fused to each other, and the drug enclosed therein is prone to leakage. This all limits the development of targeted drug delivery systems to some extent.
  • CD44 is a type of adhesion molecule that is widely distributed on the surface of lymphocytes, monocytes, endothelial cells, and the like.
  • the main ligand for the CD44 molecule is hyaluronic acid (abbreviated as "HA").
  • HA hyaluronic acid
  • CD44 Based on the activation state of the expressed cells, CD44 can be classified into a relatively static state (which cannot bind to HA), an induced activation state (which can bind to HA after activation), and a structurally active state (which can bind to HA without activation), while most normal cells
  • the CD44 of the surface is in a relatively static state and thus cannot be combined with HA.
  • CD44 is not an ideal target with significant targeting specificity. This is because CD44 is widely distributed in the human body, especially on the surface of organs rich in reticuloendothelial. Therefore, the development of a targeted drug delivery system targeting CD44 encounters the problem that if the affinity of CD44 on the surface of the target cell to HA is insufficient to provide significant specificity, then such a targeted drug delivery system will There is no specific targeting performance.
  • the inventors have found that CD44 on the surface of vulnerable plaque cells such as endothelial cells, macrophages, and smooth muscle cells is exposed to the microenvironment of vulnerable plaques (such as under the influence of inflammatory factors) compared to normal cells.
  • the induced activation results in a sudden increase in the ability to bind to HA by several tens of times.
  • This finding suggests that the presence of a large number of activated CD44 molecules on the cell surface at vulnerable plaques provides an ideal target for targeted drug delivery systems with HA as a targeting ligand.
  • the present invention provides a targeted delivery system capable of specifically targeting activated CD44 molecules, particularly targeting vulnerable plaques.
  • the present invention also provides a targeted drug delivery system capable of specifically targeting vulnerable plaque while at the same time enabling stable sustained release of the drug.
  • loading CD44 activator can promote the further activation of CD44 on the surface of the lesion cells, can amplify the targeting affinity of CD44 for HA in a short time, and significantly increase the concentration of targeting composition bound to the cell surface, which is vulnerable to The diagnosis and treatment of plaques has positive significance.
  • the targeted drug delivery system of the present invention can be loaded with a CD44 activator which can significantly increase the concentration of the tracer or therapeutic compound in a short period of time to improve diagnostic resolution or therapeutic efficacy.
  • the inventors have also found that in vulnerable plaques, with the high activation and overexpression of CD44, the endogenous macromolecular HA is also stimulated to generate a large amount, and binds to CD44 on the cell surface, promoting macrophages and lymphocytes. Aggregation within vulnerable plaques.
  • Such endogenous HA which binds to CD44 on the cell surface, forms a barrier to drug entry and reduces the bioavailability of the drug.
  • the targeted drug delivery system of the present invention can be loaded with a small molecule of hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to CD44 molecules on the cell surface at the vulnerable plaque, which The competitive binding of endogenous HA on the surface relieves the barrier formed by endogenous HA on the cell surface, which facilitates the smooth entry of the drug into the lesion cells and provides a significant therapeutic effect.
  • the present invention relates to the following aspects:
  • the present invention provides a micellar nanocarrier delivery system for targeting targeted activated CD44 molecules.
  • the present invention provides a micellar nanocarrier delivery system for targeting vulnerable plaque.
  • the invention also provides a method for preparing a nanocarrier delivery system for targeting vulnerable plaques of the invention.
  • the invention also provides a medicament comprising a nanocarrier delivery system for targeting vulnerable plaques of the invention and a pharmaceutically acceptable carrier.
  • the invention also provides a diagnostic formulation comprising a nanocarrier delivery system for targeting vulnerable plaques according to the invention.
  • the invention also provides the use of a nanocarrier delivery system for vulnerable plaques of the invention for the preparation of a medicament for the prevention and/or treatment of a vulnerable plaque or a disease associated with a vulnerable plaque.
  • the invention also provides the use of the nanocarrier delivery system of the present invention for targeting vulnerable plaque in the preparation of a diagnostic preparation for the diagnosis of a vulnerable plaque or a disease associated with a vulnerable plaque.
  • the invention also provides a method for preventing and/or treating a vulnerable plaque or a disease associated with a vulnerable plaque, the method comprising administering to a subject in need thereof a targeted vulnerable plaque according to the invention Block nanocarrier delivery system.
  • the invention also provides a method for diagnosing a vulnerable plaque or a disease associated with a vulnerable plaque, the method comprising administering to a subject in need thereof the nanocarrier targeting the vulnerable plaque of the invention Delivery system.
  • Vulnerable plaque also known as “unstable plaque” refers to atherosclerotic plaques that have a tendency to thrombosis or are likely to progress rapidly into "criminal plaques”, mainly including ruptured plaques and erosive plaques. Block and partial calcified nodular lesions. A large number of studies have shown that most of the acute myocardial infarction and stroke are caused by the rupture of vulnerable plaques with mild to moderate stenosis and secondary thrombosis.
  • Histological manifestations of vulnerable plaque include active inflammation, thin fibrous cap and large lipid core, endothelial exfoliation with surface platelet aggregation, plaque fissures or damage, and severe stenosis, as well as surface calcification, yellow luster Plaque, intraplaque hemorrhage and positive remodeling.
  • “Disease associated with vulnerable plaque” mainly refers to the “vulnerable plaque” associated with the occurrence and development of the disease, which is characterized by “vulnerable plaque”, caused by “vulnerable plaque” or secondary to “Vulnerable plaque” disease.
  • "Severe diseases associated with vulnerable plaque” mainly include atherosclerosis, coronary atherosclerotic heart disease (including acute coronary syndrome, asymptomatic myocardial ischemia - occult coronary heart disease, angina pectoris, myocardial infarction, Ischemic heart disease, sudden death, in-stent restenosis), cerebral atherosclerosis (including stroke), peripheral vascular atherosclerosis (including occlusive peripheral atherosclerosis, retinal atherosclerosis, Carotid atherosclerosis, renal atherosclerosis, lower extremity atherosclerosis, upper extremity atherosclerosis, atherosclerotic impotence), aortic dissection, hemangioma, thromboembolism, heart failure and heart
  • Targeteted drug delivery system refers to a drug delivery system that has the ability to target administration. After administration via a route, the drug contained in the targeted drug delivery system will be specifically enriched at the target site by the action of a specific carrier or targeting warhead (eg, a targeting ligand).
  • a targeting ligand e.g. a targeting ligand
  • Means currently known for achieving targeted administration include the use of passive targeting properties of various microparticle delivery systems, chemical modification on the surface of microparticle delivery systems, utilization of specific physicochemical properties, and utilization of antibody-mediated targets.
  • the "micelle nanocarrier” means a precursor polymer in which the precursor molecule of the micelle is a hydrophilic polymer having a hydrophilic structure.
  • the hydrophilic segment may be a synthetic polymer material such as polyethylene glycol, polyamino acid or hydrophilic natural polymer material (hyaluronic acid, chitosan, dextran, pullulan, alginate, heparin) Etc.); the hydrophobic segment is attached to the hydrophilic structure by polymerization (graft polymerization, block polymerization) or chemical bonding.
  • the chemical composition of the hydrophobic structure is polyphenylene ether (PPO), polycarbonate (PC), polylactic acid.
  • the polymer structure may also be a small molecule hydrophobic structure such as by bonding the hydrophobic domain: 5 ⁇ -cholic acid, 1,5-ditetradecyl-L-glutamate, dihexadecylamine Hydrophobic fragments such as ceramide.
  • the amphiphilic polymeric material facilitates self-assembly into a micellar nanocarrier in the aqueous phase and facilitates coupling of a targeting ligand on its surface.
  • Vesicles are molecularly ordered assemblies of certain amphiphilic molecules that spontaneously form a closed bilayer structure when dispersed in water, many types of nanocarriers with similar structures, or intermediates in the synthesis process.
  • the body can be called a vesicle.
  • hyaluronic acid (abbreviated as "HA") is a polymer of a polymer having the formula: (C 14 H 21 NO 11 )n. It is a higher polysaccharide consisting of the units D-glucuronic acid and N-acetylglucosamine. D-glucuronic acid and N-acetylglucosamine are linked by a ⁇ -1,3-glycosidic bond, and the disaccharide units are linked by a ⁇ -1,4-glycosidic bond.
  • Hyaluronic acid displays various important physiological functions in the body with its unique molecular structure and physicochemical properties, such as lubricating joints, regulating the permeability of blood vessel walls, regulating protein, water and electrolyte diffusion and operation, and promoting wound healing. It is especially important that hyaluronic acid has a special water retention effect and is the best moisturizing substance found in nature.
  • Derivative of hyaluronic acid refers to any derivative of hyaluronic acid capable of retaining the specific binding ability of hyaluronic acid to CD44 molecules on the surface of cells at vulnerable plaques, including but not limited to transparent
  • Judging whether a substance is a "derivative of hyaluronic acid” can be achieved by measuring the specific binding ability of the substance to the CD44 molecule on the cell surface at the vulnerable plaque, which is within the skill of those skilled in the art. Inside.
  • CD44 molecule is a type of transmembrane proteoglycan adhesion molecule widely expressed on the cell membrane of lymphocytes, monocytes, endothelial cells, etc., from the extracellular segment, the transmembrane segment and the intracellular segment.
  • the composition of the sections. CD44 molecules can mediate the interaction between a variety of cells and cells, cells and extracellular matrix, participate in the transmission of various signals in the body, and thus change the biological function of cells.
  • the primary ligand for the CD44 molecule is hyaluronic acid, and its receptor-ligand binding to hyaluronic acid determines the adhesion and/or migration of cells in the extracellular matrix.
  • CD44 molecules are also involved in the metabolism of hyaluronic acid.
  • a first aspect of the invention provides a micellar nanocarrier delivery system for targeting activated CD44 molecules, the surface of which is partially modified by a targeting ligand that is capable of The activated CD44 molecule specifically binds to the ligand.
  • a second aspect of the invention provides a micellar nanocarrier delivery system for targeting vulnerable plaque, the surface of the nanocarrier being partially modified by a targeting ligand, the targeting ligand being capable of A ligand specifically binding to the CD44 molecule on the cell surface at the vulnerable plaque.
  • the surface of the nanocarrier can also be modified to have a better effect. Modification of PEG on the surface of the carrier can play a long-circulating effect and prolong the half-life of the drug; modification of the transmembrane peptide on the surface of the carrier, self-peptide SEP, or simultaneous modification of the dual ligand can all amplify the effect of the drug.
  • micellar nanocarrier delivery system according to the first or second aspect of the invention, wherein the micellar nanocarrier is selected from the group consisting of polylactic acid/polyethylene glycol nano (PLA-PEG), polycaprolactone/polyethylene glycol Carrier (PEG-PCL).
  • PVA-PEG polylactic acid/polyethylene glycol nano
  • PEG-PCL polycaprolactone/polyethylene glycol Carrier
  • the targeting ligand is selected from the group consisting of GAG, collagen, laminin, fibronectin, selectin, osteopontin (OPN), and a monoclonal antibody HI44a, HI313, A3D8, H90, IM7, or a derivative of hyaluronic acid or a hyaluronic acid capable of specifically binding to a CD44 molecule on the cell surface at a vulnerable plaque;
  • the targeting ligand is selected from the group consisting of collagen, hyaluronic acid, selectin, osteopontin or monoclonal antibody HI44a, IM7.
  • a nanocarrier delivery system according to the first or second aspect of the invention, wherein the nanocarrier is loaded with a substance for diagnosing, preventing and/or treating a disease associated with the presence of a CD44 molecule activation condition.
  • a nanocarrier delivery system according to the first or second aspect of the invention, wherein the nanocarrier is loaded with a substance for diagnosing, preventing and/or treating a vulnerable plaque or a disease associated with a vulnerable plaque;
  • the substance is a substance for diagnosing a vulnerable plaque or a disease associated with a vulnerable plaque
  • the substance for diagnosing a vulnerable plaque or a disease associated with a vulnerable plaque is a tracer
  • the tracer is selected from the group consisting of a CT tracer, an MRI tracer, and a nucleus tracer;
  • the CT tracer is selected from the group consisting of an iodine nano contrast agent, a gold nano contrast agent, a cerium oxide nano contrast agent, a cerium nano contrast agent, a lanthanide nano contrast agent, or other similar structure tracer; More preferably, it is an iodinated contrast agent or nano gold, or other similar structure of a tracer; further preferably iohexol, iodine acid, ioversol, iodixanol, iopromide, iodobiol, iodine Poole, iopamidol, iodine, iodine, biliary acid, iodobenzoic acid, iodomate, phagoic acid, sodium iodate, iodophenyl ester, iopanoic acid, iodine Acid, sodium iodine iodate, iodine, acetophenone,
  • the MRI tracer is selected from the group consisting of a longitudinal relaxation contrast agent and a transverse relaxation contrast agent; more preferably a paramagnetic contrast agent, a ferromagnetic contrast agent and a supermagnetic contrast agent; further preferably Gd-DTPA and its line type, ring Type polyamine polycarboxylate chelate and manganese porphyrin chelate, macromolecular europium chelate, biomacromolecular modified europium chelate, folic acid modified europium chelate, dendrimer, a liposome modified developer and a tracer containing fluorene fullerene, or other similar structure; more preferably guanidinium citrate, gadolinium citrate, mussel guanamine, guanidine diamine, citric acid Ferric ammonium effervescent granules, paramagnetic iron oxide (Fe 3 O 4 NPs), or other similar structure tracer, preferably Fe 3 O 4 NPs;
  • the nuclides tracer is selected from the group consisting of carbon 14 ( 14 C), carbon 13 ( 13 C), phosphorus 32 (32P), sulfur 35 (35S), iodine 131 (131I), hydrogen 3 (3H), ⁇ 99 (99Tc), fluorine 18 (18F) labeled fluorodeoxyglucose.
  • the substance is one or more of a drug, polypeptide, nucleic acid, and cytokine for use in diagnosing, preventing, and/or treating a disease associated with a vulnerable plaque or a vulnerable plaque.
  • the substance is a CD44 activator
  • the CD44 activator is a CD44 antibody mAb or IL5, IL12, IL18, TNF-[alpha], LPS.
  • the substance is a small molecule hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to a CD44 molecule on the surface of a cell at a vulnerable plaque;
  • the small molecule hyaluronic acid or a derivative of hyaluronic acid capable of specifically binding to a CD44 molecule on the cell surface at the vulnerable plaque has a molecular weight ranging from 1 to 500 KDa, preferably 1 20 KDa, more preferably 2-10 KDa.
  • the nanocarrier is simultaneously loaded with a substance and a CD44 activator for diagnosing, preventing and/or treating a vulnerable plaque or a disease associated with a vulnerable plaque;
  • the nanocarrier is simultaneously loaded with a substance for preventing and/or treating a vulnerable plaque or a disease associated with a vulnerable plaque and hyaluronic acid or a CD44 molecule capable of interacting with a cell surface at a vulnerable plaque a specifically bound derivative of hyaluronic acid;
  • the nanocarrier is simultaneously loaded with a substance for diagnosing a vulnerable plaque or a disease associated with a vulnerable plaque, for preventing and/or treating a vulnerable plaque or a disease associated with a vulnerable plaque.
  • a substance for diagnosing a vulnerable plaque or a disease associated with a vulnerable plaque for preventing and/or treating a vulnerable plaque or a disease associated with a vulnerable plaque.
  • the substance is a substance for preventing and/or treating a vulnerable plaque or a disease associated with a vulnerable plaque
  • the substance for preventing and/or treating a vulnerable plaque or a disease associated with a vulnerable plaque is selected from the group consisting of a statin, a fibrate, an antiplatelet drug, a PCSK9 inhibitor, an anticoagulant, Angiotensin-converting enzyme inhibitors (ACEI), calcium antagonists, MMPs inhibitors, beta blockers, glucocorticoids or other anti-inflammatory substances such as the IL-1 antibody canakinumab, and their pharmaceutically acceptable
  • ACEI Angiotensin-converting enzyme inhibitors
  • calcium antagonists calcium antagonists
  • MMPs inhibitors beta blockers
  • glucocorticoids glucocorticoids
  • IL-1 antibody canakinumab glucocorticoids
  • One or more of the salts including active agents of these classes of drugs or substances, and endogenous anti-inflammatory cytokines such as interleukin 10 (IL-10);
  • the substance for preventing and/or treating a vulnerable plaque or a disease associated with a vulnerable plaque is selected from the group consisting of lovastatin, atorvastatin, rosuvastatin, simvastatin, fluvastatin Statins, pitavastatin, pravastatin, bezafibrate, ciprofibrate, clofibrate, gemfibrozil, fenofibrate, probucol, anti-PCSK9 antibodies such as evolocumab, alirocumab, bococizumab, RG7652 LY3015014 and LGT-209, or adnectin such as BMS-962476, antisense RNAi oligonucleotides such as ALN-PCSsc, nucleic acids such as microRNA-33a, microRNA-27a/b, microRNA-106b, microRNA-302, microRNA-758, microRNA -10b, microRNA-19b, microRNA-26, micro
  • a third aspect of the invention provides a method for the preparation of a nano-delivery system for targeting vulnerable plaques according to the first or second aspect, the method comprising the steps of:
  • micellar precursor molecules to diagnose, prevent, and/or treat vulnerable plaque or to vulnerable plaque
  • the substance of the disease is dissolved in a suitable organic solvent, a certain volume of aqueous solution is added, and the probe is super
  • micellar nano delivery system Sound, obtaining a drug-loaded micellar nano delivery system
  • step (3) optionally removing, by dialysis, the unloaded contained in the crude suspension obtained in the step (2) for diagnosing, preventing and/or treating vulnerable plaque or associated with vulnerable plaque
  • the substance of the disease gets a loaded nano delivery system.
  • a fourth aspect of the invention provides a medicament comprising the micellar nanocarrier delivery system of the first aspect or the second aspect, and a pharmaceutically acceptable carrier.
  • a fifth aspect of the invention provides a diagnostic preparation comprising the micellar nanocarrier delivery system of the first aspect or the second aspect.
  • a sixth aspect of the invention provides the micellar nanocarrier delivery system of the first aspect or the second aspect, the medicament of the fourth aspect, or the diagnostic preparation of the fifth aspect, which is prepared for prevention and/or Use in the treatment of a medicament for a disease associated with the activation of a CD44 molecule.
  • a seventh aspect of the invention provides the nanocarrier delivery system of the first aspect or the second aspect, the medicament of the fourth aspect, or the diagnostic preparation of the fifth aspect, which is prepared for prevention and/or treatment Use in medicinal and/or diagnostic preparations of vulnerable plaque or diseases associated with vulnerable plaque.
  • the vulnerable plaque is selected from one or more of a ruptured plaque, an aggressive plaque, and a partially calcified nodular lesion;
  • the disease associated with vulnerable plaque is selected from the group consisting of atherosclerosis, coronary atherosclerotic heart disease (including acute coronary syndrome, asymptomatic myocardial ischemia - occult coronary heart disease, angina pectoris, Myocardial infarction, ischemic heart disease, sudden death, in-stent restenosis, cerebral atherosclerosis (including stroke), peripheral vascular atherosclerosis (including occlusive peripheral atherosclerosis, retinal atherosclerosis) Sclerosis, carotid atherosclerosis, renal atherosclerosis, lower extremity atherosclerosis, upper extremity atherosclerosis, atherosclerotic impotence), aortic dissection, hemangioma, thromboembolism, heart rate One or more of failure and cardiogenic shock.
  • coronary atherosclerotic heart disease including acute coronary syndrome, asymptomatic myocardial ischemia - occult coronary heart disease, angina pectoris, Myocardial infarction
  • An eighth aspect of the invention provides a method for preventing and/or treating a disease associated with the presence of a CD44 molecule activation condition, the method comprising: administering a first aspect or a second aspect to a subject in need thereof The nanocarrier delivery system, the medicament of the fourth aspect, or the diagnostic preparation of the fifth aspect.
  • a ninth aspect of the invention provides a method for preventing, diagnosing and/or treating a vulnerable plaque or a disease associated with a vulnerable plaque, the method comprising: administering a first to a subject in need thereof
  • the nanocarrier delivery system of aspect or the second aspect, the medicament of the fourth aspect, or the diagnostic preparation of the fifth aspect comprising: administering a first to a subject in need thereof
  • the vulnerable plaque is selected from one or more of a ruptured plaque, an aggressive plaque, and a partially calcified nodular lesion.
  • the disease associated with vulnerable plaque is selected from the group consisting of atherosclerosis, coronary atherosclerotic heart disease (including acute coronary syndrome, asymptomatic myocardial ischemia - occult coronary heart disease, angina pectoris) , myocardial infarction, ischemic heart disease, sudden death, in-stent restenosis), cerebral atherosclerosis (including stroke), peripheral vascular atherosclerosis (including occlusive peripheral atherosclerosis, retinal atherosclerosis) Sclerosis, carotid atherosclerosis, renal atherosclerosis, lower extremity atherosclerosis, upper extremity atherosclerosis, atherosclerotic impotence), aortic dissection, hemangioma, thromboembolism, One or more of heart failure and cardiogenic shock.
  • coronary atherosclerotic heart disease including acute coronary syndrome, asymptomatic myocardial ischemia - occult coronary heart disease, angina pectoris
  • a tenth aspect of the invention provides a method for diagnosing a disease associated with the presence of a CD44 molecule activation condition, the method comprising: administering to a subject in need thereof the nanoparticle of the first aspect or the second aspect A carrier delivery system, the medicament of the fourth aspect, or the diagnostic preparation of the fifth aspect.
  • micellar nanocarrier delivery system of the present invention has the following advantages for diseases in which the CD44 molecule is activated:
  • micellar nanocarrier delivery system of the present invention is capable of specifically binding to an activated CD44 molecule and is capable of achieving stable sustained release of the drug.
  • CD44 in vulnerable plaques is activated by the extracellular matrix microenvironment, overexpressed in a large amount, and the affinity of CD44-HA is significantly increased, making the interaction between CD44 and HA in vulnerable plaques extremely significant. Affinity specificity.
  • CD44 within the vulnerable plaque constitutes an excellent target for the nanocarrier delivery system of the present invention to target vulnerable plaques.
  • micellar nanocarrier delivery system of the present invention for targeting vulnerable plaques is capable of actively targeting into vulnerable plaques and binding to focal cells.
  • the delivery system can achieve sustained release of the loaded material at the lesion, significantly increasing and continuously maintaining the concentration of the substance in the lesion area, thereby improving the diagnostic or therapeutic effect of the delivery system.
  • lipid pool in the vulnerable plaque, which contains a large amount of oxidized low density lipoprotein (ox-LDL). Liposomes are unstable in such an environment, and are highly disintegratable to achieve a controlled release function; whereas the nanocarrier delivery system targeting vulnerable plaques of the present invention is in vulnerable plaques.
  • the lipid pool is relatively stable and can release the drug continuously, thereby maintaining the drug concentration at the lesion.
  • the nanocarrier delivery system for targeting vulnerable plaques of the present invention may also be loaded with a CD44 activating substance, i.e., a CD44 activator such as IL5, IL12, IL18, TNF- ⁇ , LPS.
  • a CD44 activating substance i.e., IL5, IL12, IL18, TNF- ⁇ , LPS.
  • Loading CD44 activator can promote the further activation of CD44 on the surface of the lesion cells, can amplify the targeting affinity of CD44 for hyaluronic acid in a short time, and significantly increase the concentration of targeted nanocarrier composition bound to the cell surface, which is vulnerable to vulnerable spots.
  • the tracer diagnosis and treatment of the block is of positive significance because it can significantly increase the concentration of the tracer or therapeutic compound in a short period of time to improve diagnostic resolution or therapeutic effect.
  • Example 1 is a scanning electron microscope (SEM) result of a PLGA-(R)-PEG-HA micelle nanocarrier in Example 1.
  • Example 2 is an infrared characterization (FT-IR) result of the PLGA-(R)-PEG-HA micelle nanocarrier in Example 1.
  • FT-IR infrared characterization
  • Example 3 is an infrared characterization (FT-IR) result of the PLGA-(R)-PEG-SP micelle nanocarrier in Example 2.
  • Example 4 is an infrared characterization result of the PLGA-(R)-PEG-HA/Tat micelle nanocarrier in Example 3.
  • Example 5 is an infrared characterization result of the PCL-(At)-PEG-HA micelle nanocarrier in Example 4.
  • Example 6 is an infrared characterization result of the PCL-(At)-PEG-SEP/IM7 micellar nanocarrier in Example 5.
  • Figure 7 is a transmission electron microscope (TEM) image of PCL-(At)-Arg(miRNA-33a)-PEG-IM7 in Example 6.
  • Figure 8 is a transmission electron microscope (TEM) image of PLGA-(AuNP/R)-PEG-OPN in Example 7.
  • Figure 9 is a graph showing the results of infrared characterization of PLGA-(AuNP/R)-PEG-OPN in Example 7.
  • Figure 10 is a TEM result of PLGA-(Fe 3 O 4 /DXMS)-PEG-HI44a in Example 8.
  • Figure 11 is an infrared characterization result of PLGA-(Fe 3 O 4 /DXMS)-PEG-HI44a in Example 8.
  • Figure 12 is a graph showing the results of infrared characterization of PCL-(Asp/Clo)-PEG-Col in Example 9.
  • Figure 13 is a graph showing the hydrated particle size of micelle nanocarriers prepared by HA of various molecular weights in Test Example 1 as a function of storage time.
  • Figure 14 is a graph showing the change in the hydrated particle size of various types of micelle nano drug carriers in Test Example 1 with storage time.
  • Figure 15 is the effect of long-term placement in Test Example 1 on the drug encapsulation efficiency.
  • Figure 16 is a graph showing the in vitro release drug results of various micelle carriers in Test Example 1, (A) drug release rate in the first 5 hours (h); and (B) drug release rate in 120 hours.
  • Figure 17 is a nuclear magnetic resonance imaging image of a mouse atherosclerotic vulnerable plaque model constructed in Test Example 2.
  • Figure 18 is a graph showing the results of CD44 content (in semi-quantitative integration) on the surface of endothelial cells at normal arterial wall endothelial cells and arterial vulnerable plaques of model mice.
  • Figure 19 is a graph showing the results of binding of CD44 to HA on the surface of endothelial cells of normal arterial wall and arterial vulnerable plaques of model mice (indicated by binding force integral).
  • Fig. 20 is a graph showing the results of binding of CD44 to HA on the surface of macrophages in the model mouse and the macrophage surface of the arterial vulnerable plaque (indicated by binding force integral).
  • Figure 21 is a diagram showing the carotid vulnerable lesions of model mice by PLGA-(R)-PEG-HA, PLGA-(R)-PEG-SP, PLGA-(R)-PEG-HA/Tat nano delivery system of the present invention. The therapeutic effect of the block.
  • Figure 22 shows carotid artery fragility in model mice by PCL-(At)-PEG-HA, PCL-(At)-PEG-SEP/IM7, PCL-(At/miRNA-33a)-PEG-IM7 nano delivery system The therapeutic effect of the plaque.
  • Figure 23 is a graph showing the in vivo trace effect of PLGA-(AuNP/R)-PEG-OPN and other CT tracer nano delivery systems on carotid vulnerable plaques in model mice.
  • Figure 24 is a graph showing the therapeutic effect of the PLGA-(AuNP/R)-PEG-OPN nano-delivery system on carotid vulnerable plaques in model mice.
  • Figure 25 is a graph showing the in vivo trace effect of PLGA-(Fe3O4/DXMS)-PEG-HI44a and other MRI tracer nano delivery systems on carotid vulnerable plaques in model mice.
  • Figure 26 is a graph showing the therapeutic effect of the PLGA-(Fe3O4/DXMS)-PEG-HI44a nano-delivery system on carotid vulnerable plaques in model mice.
  • Figure 27 is a graph showing the therapeutic effect of the PCL-(Asp/Clo)-PEG-Col nano-delivery system on rupture of arterial vulnerable plaque in model mice.
  • This patent prepares the PLGA-PEG nanocarrier-encapsulated drug rosuvastatin (R), which utilizes the hyaluronic acid (HA) targeting molecules contained in the micelles to achieve targeted recognition and drug delivery.
  • R PLGA-PEG nanocarrier-encapsulated drug rosuvastatin
  • HA hyaluronic acid
  • Aminated PLGA-PEG (PLGA-PEG-NH2) was purchased from Xi'an Ruiqi Biotechnology Company. A certain amount of PLGA-PEG-NH2 material was dissolved in DMF to prepare 10 mg mL-1 solution, 10m PLGA-PEG solution was mixed with 40mL pure water, and probed for 3min to obtain uniform PLGA-PEG-NH2 micelle nanocarrier. .
  • PLGA-PEG-NH2 50 mg were weighed and dissolved in 50 mL of DMF, 10 m PLGA-PEG solution was mixed with 40 mL of pure water, and probed for 3 min to obtain uniform PLGA-PEG loaded with rosuvastatin.
  • -NH2 micellar nanocarrier PLGA-(R)-PEG-NH2 micellar nanocarrier
  • the carrier was purified by a 10K molecular weight cut-off ultrafiltration tube, and the loaded rosuvastatin free drug was removed to obtain a drug-loaded nanovesicle containing rosuvastatin, and the lyophilized sample was obtained as a white drug pure product.
  • HA powder having a molecular weight of 1000 D was weighed and dissolved in 10 mL of ultrapure water, and an activator EDC 25 mg, sulfo-NHS 20 mg was added, and the reaction was activated at room temperature for 30 min. After adding 90 mL of absolute ethanol to precipitate activated HA molecules (sulfo-NHS-HA), the supernatant was removed, and the precipitate was redissolved in 10 mL of water to obtain 10 mg mL-1 of a sulfo-NHS-HA solution.
  • the prepared PLGA-(R)-PEG-NH2 lyophilized powder was dissolved in 5 mM NaHCO3 to prepare a 10 mg mL-1 solution, and 10 mL of the solution was added to 1.0 mL of 10 mg mL-1 sulfo-NHS-HA at room temperature.
  • the reaction was stirred for 24 hours, and the carrier was purified by a 100 K molecular weight cut-off ultrafiltration tube to remove unconjugated sulfo-NHS-HA, thereby obtaining targeted drug-loaded nanovesicles linked to the target molecule HA and entrapped rosuvastatin.
  • the lyophilized sample was obtained as a pure white drug (PLGA-(R)-PEG-HA).
  • micellar nanocarriers can be prepared by a similar method using different molecular weights of HA (1-500 KDa).
  • Figure 1 shows the results of scanning electron microscopy (SEM) of PLGA-(R)-PEG-HA micellar nanocarriers.
  • Figure 2 shows the results of infrared characterization (FT-IR) of PLGA-(R)-PEG-HA micellar nanocarriers.
  • Aminated PLGA-PEG (PLGA-PEG-NH 2 ) was purchased from Xi'an Ruiqi Biotechnology Co., Ltd., and 50 mg of PLGA-PEG and 50 mg of rosuvastatin (R) were weighed together in 10 mL of DMF, and 10 mL of PLGA-PEG was added. The solution was mixed with 40 mL of pure water and ultrasonically loaded with probe for 3 min to obtain a uniform micellar nanocarrier (PLGA-(R)-PEG) loaded with rosuvastatin. The carrier was purified by a 10K molecular weight cut-off ultrafiltration tube to remove the unencapsulated free drug, and the R-loaded drug-loaded nanomicelle was obtained, and the lyophilized sample was obtained to obtain a white drug pure product.
  • the prepared PLGA-(R)-PEG lyophilized powder was dissolved in 5 mM NaHCO 3 to prepare a 10 mg mL -1 solution, and 10 mL of the solution was added to 1.0 mL of 1 mg mL -1 sulfo-NHS-SP, and stirred at room temperature. After reacting for 4 hours, the nano drug was purified by Sephadex column G-100 to remove the unconjugated sulfo-NHS-SP, and the targeted drug-loaded nano-gel connected with the target molecule SP and entrapped rosuvastatin was obtained. Bundle, lyophilized sample to obtain white drug pure product (PLGA-(R)-PEG-SP)
  • Figure 3 is the infrared characterization (FT-IR) result of PLGA-(R)-PEG-SP micelle nanocarrier.
  • This patent prepares PLGA-(R)-PEG-HA/Tat nanomicelle-encapsulated drug rosuvastatin (R), nanomicelle itself contains hyaluronic acid HA and transmembrane peptide Tat, double modification in significantly increasing nanometer
  • the targeted enrichment ability of the vector greatly enhances the effect of targeted recognition and drug delivery.
  • Aminated PLGA-PEG (PLGA-PEG-NH 2 ) was purchased from Xi'an Ruiqi Biotechnology Co., Ltd., and 50 mg of PLGA-PEG and 50 mg of rosuvastatin (R) were weighed together in 50 mL of DMF, and 10 mg of PLGA-PEG was added. The solution was mixed with 40 mL of pure water, and ultrasonically loaded with a probe for 3 min to obtain a uniform micellar nanocarrier (PLGA-(R)-PEG-NH2) loaded with R. The carrier was purified by a 10K molecular weight cut-off ultrafiltration tube to remove the unencapsulated free drug, thereby obtaining R-coated micelle nanocarrier, and lyophilizing the sample to obtain a white drug pure product.
  • the prepared PLGA-(R)-PEG lyophilized powder was dissolved in 5 mM NaHCO 3 to prepare a 10 mg mL-1 solution, and 10 mL of the solution was added to 0.5 mL of 1 mg mL-1 sulfo-NHS-HA, and 0.5.
  • the 1 mg mL-1 sulfo-NHS-Tat in mL was stirred for 4 hours at room temperature, and the nano drug was purified by G-100 Sephadex column to remove the unconjugated targeting molecule to obtain the linked targeting molecule and entrapped.
  • the patent preparation of the PCL-PEG micelle nanocarrier entrapped drug atorvastatin (At) utilizes hyaluronic acid (HA) targeting molecules contained in the micelle nanocarrier itself to achieve targeted recognition and drug delivery.
  • HA hyaluronic acid
  • PCL-PEG-NH2 Aminated PCL-PEG
  • HA powder having a molecular weight of 30,000 was weighed and dissolved in 10 mL of ultrapure water, and an activator EDC 25 mg, sulfo-NHS 20 mg was added, and the reaction was activated at room temperature for 30 min. After adding 90 mL of absolute ethanol to precipitate activated HA molecules (sulfo-NHS-HA), the supernatant was removed, and the precipitate was redissolved in 10 mL of water to obtain 10 mg mL-1 of a sulfo-NHS-HA solution.
  • the prepared PCL-(At)-PEG-NH2 lyophilized powder was dissolved in 5 mM NaHCO3 to prepare a 10 mg mL-1 solution, and 10 mL of the solution was added to 1.0 mL of 10 mg mL-1 sulfo-NHS-HA at room temperature.
  • the reaction was stirred for 24 hours, and the carrier was purified by a 100K molecular weight cut-off ultrafiltration tube to remove the unconjugated sulfo-NHS-HA, and the targeted micellar nanocarrier which is linked to the targeted molecule HA and entrapped atorvastatin was obtained, and frozen.
  • the dry sample was obtained as a pure white drug (PCL-(At)-PEG-HA).
  • Figure 5 shows the results of infrared characterization of PCL-(At)-PEG-HA.
  • PCL-PEG-NH2 Aminated PCL-PEG
  • PCL-PEG-NH2 was purchased from Xi'an Ruiqi Biotechnology Co., Ltd., and 50 mg of PCL-PEG and 50 mg of atorvastatin (At) were weighed together in 50 mL of DMF, and 10 mg of PCL-PEG solution was added.
  • the At-loaded homogeneous micellar nanocarrier (PCL-(At)-PEG) was obtained by mixing with 40 mL of pure water and ultrasonically using a probe for 3 min.
  • the carrier was purified by a 10K molecular weight cut-off ultrafiltration tube to remove the unencapsulated free drug, and the At-loaded drug-loaded nanomicelle was obtained, and the lyophilized sample was obtained to obtain a white drug pure product.
  • IM7 1.0 mg of IM7 in 1 mL of ultrapure water, add 0.5 mg of activator EDC, 0.5 mg of sulfo-NHS, and activate the reaction at room temperature for 30 min.
  • the activated IM7 was purified by a 10K molecular weight cut-off ultrafiltration tube, and the small molecule activator precipitate was removed and dissolved in 1.0 mL of PBS buffer solution to obtain a 1 mg mL-1 sulfo-NHS-IM7 solution.
  • the prepared PCL-(At)-PEG lyophilized powder was dissolved in 5 mM NaHCO3 to prepare a 10 mg mL-1 solution, and 10 mL of the solution was added to 1 mL of 1 mg mL-1 sulfo-NHS-IM7, and 0.5 mL. 1 mg mL-1 of sulfo-NHS-SEP was stirred at room temperature for 4 hours, and the nano drug was purified by G-100 Sephadex column to remove unconjugated sulfo-NHS-IM7 and sulfo-NHS-SEP to obtain a ligation target.
  • PCL-(At)-PEG-SEP/IM7 Targeted drug-loaded nanomicelles of atorvastatin, which were molecularly and entrapped, were lyophilized to obtain a pure white drug (PCL-(At)-PEG-SEP/IM7).
  • Figure 6 shows the results of infrared characterization of PCL-(At)-PEG-SEP/IM7. If it is not necessary to modify the SEP, the above-mentioned SEP-added ring can be saved to obtain PCL-(At)-PEG-IM7 without unmodified SEP.
  • This patent prepares PCL-Arg-PEG nanomicelle-encapsulated drug atorvastatin microRNA miRNA-33a, and uses nano-micelle-coupled IM7 targeting molecule to achieve targeted drug delivery.
  • PCL-Arg-PEG-NH2 was purchased from Xi'an Ruiqi Biotechnology Co., Ltd. as a block copolymer of PCL polyarginine and PEG. A certain amount of PCL-Arg-PEG material was dissolved in DMF to make 10mg mL-1 solution, 10m PCL-Arg-PEG solution was mixed with 40mL pure water, and ultrasonically probed for 3min to obtain uniform blank PCL-Arg-PEG glue. Bunch of nanocarriers.
  • PCL-Arg-PEG-NH2 and 50 mg of atorvastatin (At) were weighed and dissolved in 50 mL of DMF, 10 m of PCL-Arg-PEG-NH2 solution was mixed with 40 mL of pure water, and the probe was ultrasonicated for 3 min to obtain a load.
  • the carrier was purified by a 10K molecular weight cut-off ultrafiltration tube, and the unencapsulated free drug was removed to obtain a drug-loaded nanomicelle containing atorvastatin, and the lyophilized sample was obtained as a white drug pure product.
  • the prepared atorvastatin-loaded nanocarrier lyophilized powder was dissolved in 10 mL of 5 mM NaHCO3 to prepare a 10 mg mL-1 solution, and the miRNA-33a lyophilized powder was dissolved and added to the nanocarrier solution, and reacted at room temperature for 2 h. .
  • the vector was purified by a 10K molecular weight cut-off ultrafiltration tube to remove unbound miRNA-33a, and the drug-loaded nanomicelles containing At/miRNA33-a were simultaneously obtained, and the white drug pure product was obtained by lyophilizing the sample.
  • IM7 1 mg was weighed and dissolved in 1 mL of ultrapure water, and the activator EDC 1.0 mg, sulfo-NHS 1.0 mg was added, and the reaction was activated at room temperature for 30 min.
  • the activated IM7 was purified by a 10K molecular weight cut-off ultrafiltration tube, and the small molecule activator precipitate was removed and dissolved in 1.0 mL of PBS buffer solution to obtain a 1 mg mL-1 sulfo-NHS-IM7 solution.
  • the prepared PCL-(At/miRNA-33)-PEG lyophilized powder was dissolved in 5 mM NaHCO3 to prepare a 10 mg mL-1 solution, and 10 mL of the solution was added to 1.0 mL of 1 mg mL-1 sulfo-NHS-IM7.
  • the reaction was stirred at room temperature for 4 hours, and the nano drug was purified by G-100 Sephadex column to remove the unconjugated sulfo-NHS-IM7, and the targeted carrier of atorvastatin linked to the targeting molecule IM7 was obtained.
  • the drug nano-micelles were lyophilized to obtain a pure white drug (PCL-(At/miRNA-33a)-PEG-IM7).
  • Figure 7 is a transmission electron microscope (TEM) image of PCL-(At/miRNA-33a)-PEG-IM7.
  • This patent prepares PLGA-(AuNP/R)-PEG-OPN nanomicelle-encapsulated drug rosuvastatin (R) and gold nanoparticles (AuNPs), which are targeted by osteopontin (OPN) contained in nanomicelle itself.
  • the molecules achieve targeted recognition and drug delivery, and the AuNPs contained therein have both CT imaging functions.
  • TA-AuNPs hydrophobic lipoic acid gold nanoparticles
  • the carrier was purified by a 100K molecular weight cut-off ultrafiltration tube, and the unencapsulated free drug and TA-AuNPs were removed to obtain a drug-loaded nanomicelle containing rosuvastatin, and the sample was freeze-dried to obtain a pure drug product.
  • OPN OPN 1.0 mg was dissolved in 1 mL of ultrapure water, and the activator EDC 1.0 mg, sulfo-NHS 1.0 mg was added, and the reaction was activated at room temperature for 30 min.
  • the activated OPN was purified by a 10K molecular weight cut-off ultrafiltration tube, and the small molecule activator precipitate was removed and dissolved in 1.0 mL of PBS buffer solution to obtain a 1 mg mL-1 sulfo-NHS-OPN solution.
  • the prepared PLGA-(AuNP/R)-PEG lyophilized powder was dissolved in 5 mM NaHCO3 to prepare a 10 mg mL-1 solution, and 10 mL of the solution was added to 1.0 mL of 1 mg mL-1 sulfo-NHS-OPN at room temperature. The reaction was stirred for 4 hours, and the nano drug was purified by G-100 Sephadex column to remove unconjugated sulfo-NHS-OPN, and the targeted drug-loaded nanometer of rosuvastatin linked to the targeting molecule OPN was obtained. The micelles were lyophilized to obtain a pure white drug (PLGA-(AuNP/R)-PEG-OPN).
  • Figure 8 is a PLGA-(AuNP/R)-PEG-OPN transmission electron microscope (TEM) image.
  • Figure 9 shows the results of infrared characterization of PLGA-(AuNP/R)-PEG-OPN.
  • This patent prepares PLGA-(Fe 3 O 4 /DXMS)-PEG-HI44a nanomicelle-encapsulated drug dexamethasone (DXMS), which utilizes the monoclonal antibody (HI44a) targeting molecule coupled to the nanomicelle itself to achieve the target.
  • DXMS monoclonal antibody
  • loaded Fe3O4 NPs achieve MRI imaging while achieving therapeutic effects.
  • Fe3O4 NPs paramagnetic iron oxide coated with oleic acid was obtained.
  • the material was purified by an external magnetic field, vacuum-dried to obtain a magnetic material powder, and redispersed in DMF to obtain different concentrations of Fe3O4 NPs dispersion.
  • the carrier was purified by a 100K molecular weight cut-off ultrafiltration tube to remove the unencapsulated DXMS free drug and Fe3O4 NPs, and the DXMS-loaded drug-loaded nanomicelles were obtained, and the lyophilized sample was obtained to obtain a pure drug product.
  • HI44a 1.0 mg was weighed and dissolved in 1 mL of ultrapure water, and the activator EDC 1.0 mg, sulfo-NHS 1.0 mg was added, and the reaction was activated at room temperature for 30 min.
  • the activated HI44a was purified by a 10K molecular weight cut-off ultrafiltration tube, and the small molecule activator precipitate was removed and dissolved in 1.0 mL of PBS buffer solution to obtain 1 mg mL-1 sulfo-NHS-HI44a solution.
  • the prepared PLGA-(Fe3O4/DXMS)-PEG lyophilized powder was dissolved in 5 mM NaHCO3 to prepare a 10 mg mL-1 solution, and 10 mL of the solution was added to 1.0 mL of 1 mg mL-1 sulfo-NHS-HI44a at room temperature.
  • the reaction was stirred for 4 hours, and the nano drug was purified by G-100 Sephadex column to remove the unconjugated sulfo-NHS-HI44a, and the targeted drug-loaded nano-gel was obtained by linking the targeting molecule HI44a and encapsulating Fe3O4NPs and DXMS.
  • the bundle is lyophilized to obtain a pure drug product.
  • Figure 10 shows the TEM results of PLGA-(Fe3O4/DXMS)-PEG-HI44a.
  • Figure 11 shows the results of infrared characterization of PLGA-(Fe3O4/DXMS)-PEG-HI44a.
  • PCL-PEG-NH2 weigh 50 mg of PCL-PEG-NH2 and 20 mg of aspirin (Asp) 30 mg of clopidogrel (Clo) together in 10 mL of DMF, mix 10 m of PCL-PEG-NH2 solution with 40 mL of pure water, and use a probe for 3 min to obtain a load.
  • the vector was purified by a 10K molecular weight cut-off ultrafiltration tube, removed to entrap the free drug, and the sample was lyophilized to obtain a white drug pure product.
  • the prepared PCL-(Asp/Clo)-PEG lyophilized powder was dissolved in 5 mM NaHCO3 to prepare a 10 mg mL-1 solution, and 10 mL of the solution was added to 1.0 mL of 10 mg mL-1 sulfo-NHS-Col at room temperature.
  • the reaction was stirred for 24 hours, the carrier was purified by a 100 K molecular weight cut-off ultrafiltration tube, the unconjugated sulfo-NHS-Col was removed, and the targeted nanomicelles of the drug linked to the targeting molecule Col and entrapped were obtained, and the sample was freeze-dried to obtain white. Pure drug (PCL-(Asp/Clo)-PEG-Col).
  • Figure 12 is the results of infrared characterization of (PCL-(Asp/Clo)-PEG-Col.
  • the micelle nano delivery system loaded with the therapeutic agent prepared in Example 1 is taken as an example to prove that the delivery system of the present invention has stable and controllable properties, thereby being suitable for vulnerable plaque or vulnerable spots. Diagnosis, prevention and treatment of block-related diseases.
  • the carrier drug rosuvastatin, atorvastatin, dexamethasone, aspirin, and clopidogrel have strong UV absorption properties and can therefore be obtained by HPLC-UV method (using Waters 2487, Waters Corporation). US) Determination of the content of atorvastatin, atorvastatin, dexamethasone, aspirin, and clopidogrel by UV absorption characteristics. A standard quantitative equation was established for the peak area (Y) of the HPLC chromatographic peaks with different concentrations of rosuvastatin, atorvastatin, dexamethasone, aspirin, and clopidogrel solutions (X).
  • the drug content in the resulting liquid was measured by HPLC (Waters 2487, Waters Corporation, USA), and the encapsulation efficiency was calculated by Formula 1.
  • the nano delivery system of the present invention PLGA-(R)-PEG-HA, PLGA-(R)-PEG-SP, PLGA-(R)-PEG-HA/Tat, PCL-(At)-PEG-HA, PCL -(At)-PEG-SEP/IM7, PCL-(At/miRNA-33a)-PEG-IM7, PLGA-(AuNP/R)-PEG-OPN, PLGA-(Fe 3 O 4 /DXMS)-PEG- HI44a, PCL-(Asp/Clo)-PEG-Col were stored at 4 ° C, sampled at different time points, and passed through a laser particle size analyzer (BI-Zeta Plus/90 Plus, Brookhaven Instruments Corporation, USA) The change in the hydrated particle size was examined.
  • Figure 13 shows the hydrated particle size of the micellar nanocarrier prepared by HA of various molecular weights designed according to the present patent as a function of storage time.
  • Figure 14 shows the hydrated particle size of various micellar nano drug carriers designed according to the patent as a function of storage time.
  • micellar delivery system PLGA-(R)-PEG-HA, PLGA-(R)-PEG-SP, PLGA-(R)-PEG-HA/Tat, PCL- (At)-PEG-HA, PCL-(At)-PEG-SEP/IM7, PCL-(At/miRNA-33a)-PEG-IM7, PLGA-(AuNP/R)-PEG-OPN, PLGA-(Fe 3 O 4 /DXMS)-PEG-HI44a, PCL-(Asp/Clo)-PEG-Col drug encapsulation rate decreased from the initial 72% to 86% to 46-53%.
  • the micelle structure can better protect the internal molecules from being leaked. From the above data, it can be sufficiently explained that the long-term storage stability of the micelle delivery system of the present invention is good, and the particle size does not change much after storage at 4 ° C for two months and the leakage rate of the drug is low.
  • PLGA-(R)-PEG-HA Take 2 mL of the nano delivery system of the invention PLGA-(R)-PEG-HA, PLGA-(R)-PEG-SP, PLGA-(R)-PEG-HA/Tat, PCL-(At)-PEG-HA, PCL-(At)-PEG-SEP/IM7, PCL-(At/miRNA-33a)-PEG-IM7, PLGA-(AuNP/R)-PEG-OPN, PLGA-(Fe 3 O 4 /DXMS)-PEG - HI44a, PCL-(Asp/Clo)-PEG-Col is sealed in a dialysis bag.
  • the drug content in the release solution was measured by HPLC (Waters 2487, Waters Corporation, USA), and the cumulative release rate of the drug was calculated by Formula 3.
  • Equation 3 The meaning of each parameter in Equation 3 is as follows:
  • V e displacement volume of the release liquid, where V e is 2 mL
  • V 0 volume of the release liquid in the release system, where V 0 is 50 mL
  • C i concentration of the drug in the release liquid at the time of the i-th replacement sampling, in units of ⁇ g/mL
  • M drug the total mass of the drug in the delivery system, in ⁇ g
  • Cn drug concentration in the release system measured after the nth replacement of the release solution.
  • FIG. 16 is a graph showing changes in drug cumulative release rate of the micellar delivery system of the present invention.
  • the various types of micelle delivery systems release faster in the first 5 hours, and the 5-hour drug release rate is in the range of 19% to 34%. After that, the drug release rate gradually slowed down, and 60% to 85% of the drug was released after 60 hours.
  • the faster release rate in the early stage may be caused by the release behavior of the partially adsorbed or precipitated micelle surface which can be rapidly dissolved and diffused into the release medium, and the later drug release is mainly encapsulated in the micelle.
  • Drug release manifested as sustained, slow release behavior.
  • the results of in vitro release experiments showed that the release of the drug from the micelles can be effectively retarded.
  • the in vitro release experiments indicate that the micelles have slow and sustained release characteristics as drug carriers.
  • Figure 16 shows the in vitro drug release results for various micelle carriers, (A) drug release rate for the first 5 hours (h); (B) drug release rate for 120 hours.
  • the rosuvastatin-loaded nano delivery system prepared in Example 1 was taken as an example to demonstrate that the delivery system of the present invention can remain relatively stable at vulnerable plaques compared to liposome delivery systems. In order to achieve long-term sustained release of the drug.
  • mice SPF-grade ApoE-/- mice (18, 10 weeks old, body weight 20 ⁇ 1 g) were taken as experimental animals.
  • the mice were given an adaptive high-fat diet (fat 10% (w/w), cholesterol 2% (w/w), sodium cholate 0.5% (w/w), and the rest were normal feed for mice) after 4 weeks of feeding.
  • Anesthesia was intraperitoneally injected with 1% sodium pentobarbital (prepared by adding 1 mg of sodium pentobarbital to 100 ml of physiological saline) at a dose of 40 mg/kg.
  • the mouse was fixed on the surgical plate in the supine position, disinfected with the neck centered with 75% (v/v) alcohol, the neck skin was cut longitudinally, and the anterior cervical gland was bluntly separated, on the left side of the trachea.
  • the left common carotid artery can be seen on the side. Carefully separate the common carotid artery to the bifurcation.
  • the silicone tube sleeve with a length of 2.5 mm and an inner diameter of 0.3 mm was placed on the outer circumference of the left common carotid artery.
  • the proximal and distal centripet segments of the cannula were narrowed and fixed by filaments. .
  • LPS lipopolysaccharide
  • mice were placed in a 50 ml syringe (sufficient venting reserved) to cause restrictive mental stress, 6 hours/day, 5 days per week for a total of 6 weeks.
  • the mouse atherosclerotic vulnerable plaque model was completed at 14 weeks postoperatively.
  • Figure 17 (a) and (b) show the MRI image of the mouse atherosclerotic vulnerable plaque model. It can be seen from the arrow pointing part that the left carotid plaque has formed. Successful modeling, right arterial artery can be compared as normal arterial wall.
  • the percentage of drug exposure at the vulnerable plaque of the artery was determined by liquid chromatography-mass spectrometry (which reflects the concentration of rosuvastatin at the vulnerable plaque after injection of the test drug over time):
  • rosuvastatin 0.0141g placed in a 25mL volumetric flask, dissolved in methanol and diluted to the mark, shaken, prepared into a concentration of 56.4 ⁇ g / mL of rosuvastatin reference stock; rosulva
  • the statin reference stock solution was diluted with methanol to a series of standard solutions of 10, 1, 0.5, 0.125, 0.05, 0.025, 0.01, 0.002, 0.0004 ⁇ g/mL, and stored at 4 ° C for use.
  • the animals were sacrificed (one mouse at each time point) before and 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 168 h (seven days) before administration, and the carotid plaques were quickly removed for physiological placement.
  • the surface water was dried by a filter paper, and each was cut 1 cm, and the wet weight was weighed, and 1 ml of physiological saline was added to homogenize to prepare a homogenate.
  • 1 ml of the homogenate was taken, and 20 ⁇ L of methanol, 100 ⁇ L of an internal standard solution having a concentration of 15.2 ng/mL, 100 ⁇ L of a 10% (v/v) formic acid aqueous solution, and 5 mL of ethyl acetate were added thereto, and the mixture was mixed and centrifuged at 14,000 rpm for 10 minutes. 4 ml of the organic layer solution was taken and dried with nitrogen.
  • Rosuvastatin calcium mimics drug-containing plasma samples. Operate according to plasma treatment (add 50 ⁇ L of 15.2 ng/mL internal standard solution, 50 ⁇ L of 10% (v/v) formic acid aqueous solution, 2.5 mL of ethyl acetate, mix, centrifuge at 14000 rpm for 10 min, take 2 ml of organic layer solution, and blow with nitrogen.
  • the liquid phase separation was carried out using a Shimadzu modular LC system (Tokyo, Japan) system comprising: 1 DGU-20A3R vacuum degasser, 2 LC-20 ADXR solvent delivery modules, 1 SIL-20ACXR autosampler, 1 SPD-M20A PDA system and 1 CBM-20A controller.
  • the liquid phase system was connected in-line with an ABSciex 5500 Qtrap mass spectrometer (Foster City, CA, USA) equipped with an ESI interface. Analyst software (Version 1.6.2, ABSciex) is used for data acquisition and processing.
  • Chromatographic analysis was performed using a CortecsTM UPLC C18 column (150 mm x 2.1 mm internal diameter (i.d.), 1.6 ⁇ m particle size) (Waters Corporation, USA), and the column temperature and sample chamber temperatures were set to 40 ° C and 4 ° C, respectively.
  • the mobile phase was a 0.1% (v/v) aqueous solution of formic acid and acetonitrile (40:60, v/v) in an amount of 2 ⁇ l.
  • the flow rate was 0.2 mL/min and the analysis time for a single sample was 4 min.
  • the ion source used for mass spectrometry is the ESI source, positive ion scan mode.
  • the spray voltage was set to 4500V and the source temperature was set to 500 °C.
  • Each compound was detected by multiple reaction monitoring (MRM).
  • the ion channels of each component were: rosuvastatin m/z 482.2 ⁇ 258.2, acetaminophen m/z 152.2 ⁇ 110.
  • the collision energy and cone voltage of each compound were optimized: rosuvastatin 43V and 100V, acetaminophen 23V and 100V.
  • the retention times of rosuvastatin calcium and acetaminophen were 2.07 min and 1.49 min, respectively.
  • the density of CD44 on the surface of vulnerable plaque endothelial cells and the affinity with HA are studied to select CD44 in the vulnerable plaque as the targeted vulnerable plaque according to the present invention.
  • the target of the delivery system of the block provides an experimental basis.
  • a mouse atherosclerotic vulnerable plaque model was constructed according to the method described in Example 4 above.
  • the endothelial cells of the normal arterial vascular endothelial cells and arterial vulnerable plaques of the model mice were taken for CD44 determination by immunohistochemical staining and image analysis.
  • the specific experimental methods are as follows:
  • the mouse carotid atherosclerotic vulnerable plaque specimens were fixed with 10 mL/L formaldehyde solution, paraffin-embedded, 4 ⁇ m sections, conventional dewaxing, hydration treatment, and avidin-biotin-enzyme complex method. (SABC) detects CD44 content.
  • the specimen was immersed in a 30 mL/L H2O2 aqueous solution to block the activity of endogenous peroxidase, and subjected to antigen microwave repair in a citrate buffer. Then 50 g/L bovine serum albumin (BSA) blocking solution was added dropwise and allowed to stand at room temperature for 20 min.
  • BSA bovine serum albumin
  • a murine anti-CD44 polyclonal antibody (1:100) was added dropwise, placed in a refrigerator at 4 ° C overnight, and incubated at 37 ° C for 1 h. After washing, biotinylated goat anti-mouse IgG was added dropwise and reacted at 37 ° C for 30 min. Then, it was washed with phosphate buffered saline (PBS), and horseradish peroxidase-labeled SABC complex was added dropwise, and incubated at 37 ° C for 20 min; each step was washed with PBS. Finally, color development was performed with DAB (controlled under a microscope when developing color), followed by counterstaining, dehydration and mounting with hematoxylin.
  • PBS phosphate buffered saline
  • Sections were analyzed by immunohistochemical analysis system of BI-2000 image analysis system. Three sections were collected for normal arterial endothelial cells and endothelial cells of arterial vulnerable plaques, and five representative fields were randomly selected.
  • the positive expression of CD44 was: cell membrane, cytoplasm was brownish/tanose and the background was clear, and the darker the color, the stronger the expression of CD44. No brown-yellow particles were found to be negative for CD44 expression.
  • the mean absorbance (A) values of positive cells in the endothelial cells of normal arterial endothelial cells and arterial vulnerable plaques were measured and compared. The result is shown in Fig. 18.
  • Figure 18 shows the results of surface CD44 content determination (in semi-quantitative integration) of endothelial cells at normal arterial wall endothelial cells and arterial vulnerable plaques of model mice. As shown, the surface CD44 content of endothelial cells at arterial vulnerable plaques was approximately 2.3 times that of normal arterial endothelial cells.
  • Natural ligands for CD44 include: HA, GAG, collagen, laminin, fibronectin, selectin, osteopontin (OPN), and monoclonal antibodies HI44a, HI313, A3D8, H90, IM7, and the like.
  • the normal arterial wall endothelial cells of the model mice and the endothelial cells at the vulnerable plaques of the arteries were added with a ligand/antibody labeled with aminofluorescein at a concentration of 10 mg/ml, and the improved Iggar medium was used in Durbreco. (DMEM) medium (containing 10% by volume of calf serum, 100 U/ml penicillin, 100 U/ml streptomycin) was cultured in a 37 ° C, 5% CO 2 incubator. After 30 minutes, the mean fluorescence intensity (MFI) was determined by flow cytometry (CytoFLEX, Beckman Coulter, USA), and the binding integral of FL-ligand/antibody on both cell surfaces was calculated (to normal arterial vessels). The binding of CD44 to the ligand/antibody of the wall endothelial cells is 1). The result is shown in FIG.
  • the binding force of CD44 to HA on the surface of endothelial cells at the vulnerable plaque of arteries was approximately 24 times that of the endothelial cell surface of normal arterial wall. This indicates that most of the CD44 on the surface of endothelial cells of normal arterial wall is in a quiescent state that cannot bind to ligand HA, and CD44 on the surface of endothelial cells at the vulnerable plaque of arteries is activated by factors such as inflammatory factors in the internal environment. The affinity with HA has increased significantly.
  • CD44 other ligands similar to HA, the binding capacity of CD44 and GAG on the surface of vulnerable plaque endothelial cells is 22 times that of normal cells, and the binding capacity of CD44 and collagen in vulnerable plaque endothelial cells is 21 times that of normal cells.
  • the binding capacity of CD44 and laminin in vulnerable plaque endothelial cells is 16 times that of normal cells.
  • the binding force of CD44 and fibronectin in vulnerable plaque endothelial cells is 18 times that of normal cells, and vulnerable spots are vulnerable.
  • the binding strength of CD44 to the selectin protein was 19 times that of normal cells, and the binding capacity of CD44 and osteopontin in vulnerable plaque endothelial cells was 17 times that of normal cells.
  • CD44 monoclonal antibodies Similar results were observed for CD44 monoclonal antibodies: the binding of CD44 to H144a on the surface of vulnerable plaque endothelial cells was 15 times that of normal cells, and the binding capacity of CD44 and H1313 in vulnerable plaque endothelial cells was 21 times that of normal cells. The binding capacity of CD44 and A3D8 in vulnerable plaque endothelial cells is 17 times that of normal cells. The binding capacity of CD44 and H90 in vulnerable plaque endothelial cells is 9 times that of normal cells, and vulnerable plaque endothelial cells CD44 and IM7. The binding force integral is 8 times that of normal cells.
  • the macrophages in the peritoneal cavity of the model mice and the macrophages in the vulnerable plaques of the arteries were added to the ligand/antibody labeled with aminofluorescein at a concentration of 10 mg/ml, and the volume fraction was 10% calf serum, 100 U/ml penicillin, 100 U/ml streptomycin) were cultured in a 37 ° C, 5% CO 2 incubator. After 30 minutes, the mean fluorescence intensity (MFI) was determined using a flow cytometer (CytoFLEX, Beckman Coulter, USA) and the binding integral of FL-HA on both cell surfaces was calculated (with extra-plaque macrophages) The CD44 on the cell surface has a ligand/antibody affinity of 1). The result is shown in FIG.
  • the binding force of CD44-HA on the surface of macrophages in arterial vulnerable plaques was about 40 times that of CD44-HA on the surface of macrophage outside the plaque. This indicates that CD44 on the surface of macrophages in arterial vulnerable plaques is also activated by factors such as inflammatory factors in the internal environment, and the affinity with HA is significantly increased.
  • CD44 other ligands similar to HA, the binding capacity of CD44 and GAG on the surface of vulnerable plaque macrophages is 33 times that of normal cells, and the binding integral of CD44 and collagen in vulnerable plaque macrophages is normal cells. 38 times, the binding capacity of CD44 and laminin in vulnerable plaque macrophages is 37 times that of normal cells, and the binding force of CD44 and fibronectin in vulnerable plaque macrophages is 35 times that of normal cells.
  • the binding capacity of vulnerable plaque macrophage CD44 to selectin is 33 times that of normal cells, and the binding capacity of vulnerable plaque macrophage CD44 to osteopontin is 33 times that of normal cells.
  • CD44 monoclonal antibodies Similar results were observed for CD44 monoclonal antibodies: the binding of CD44 to H144a on the surface of vulnerable plaque macrophages was 17 times that of normal cells, and the binding integral of vulnerable plaque macrophages CD44 and H1313 was normal cells. 20 times, the binding strength of CD44 and A3D8 in vulnerable plaque macrophages is 16 times that of normal cells. The binding force of CD44 and H90 in vulnerable plaque macrophages is 9 times that of normal cells, and vulnerable plaques are giant. The binding capacity of phagocytic CD44 to IM7 is 10 times that of normal cells.
  • Test Example 4 PLGA-(R)-PEG-HA, PLGA-(R)-PEG-SP, PLGA-(R)-PEG-HA/Tat rosuvastatin delivery system of the present invention for arterial vulnerable plaque In vivo experiment
  • Hyaluronic acid (HA) and selectin (SP) are ligands for CD44, which can target vulnerable plaques.
  • Rosuvastatin (R) has the effect of reversing plaque, transmembrane peptide (Tat) Can increase local penetration and aggregation of drugs.
  • the purpose of this example was to verify that the PLGA-(R)-PEG-HA, PLGA-(R)-PEG-SP, PLGA-(R)-PEG-HA/Tat carrier delivery system of the present invention is vulnerable to arterial arteries. The in vivo therapeutic effect of plaque.
  • a physiological saline solution of free rosuvastatin was formulated, and a micelle-loaded delivery system loaded with a therapeutic agent was prepared by the method described in the above Examples 1-3.
  • SPF-grade ApoE-/- mice 42 animals, 5-6 weeks old, body weight 20 ⁇ 1 g were taken as experimental animals.
  • the mice were given an adaptive high-fat diet (fat 10% (w/w), cholesterol 2% (w/w), sodium cholate 0.5% (w/w), and the rest were normal feed for mice) after 4 weeks of feeding.
  • Anesthesia was intraperitoneally injected with 1% sodium pentobarbital (prepared by adding 1 mg of sodium pentobarbital to 100 ml of physiological saline) at a dose of 40 mg/kg.
  • the mouse was fixed on the surgical plate in the supine position, disinfected with the neck centered with 75% (v/v) alcohol, the neck skin was cut longitudinally, and the anterior cervical gland was bluntly separated, on the left side of the trachea.
  • the left common carotid artery can be seen on the side. Carefully separate the common carotid artery to the bifurcation.
  • the silicone tube sleeve with a length of 2.5 mm and an inner diameter of 0.3 mm was placed on the outer circumference of the left common carotid artery.
  • the proximal and distal centripet segments of the cannula were narrowed and fixed by filaments. .
  • LPS lipopolysaccharide
  • mice were placed in a 50 ml syringe (sufficient venting reserved) to cause restrictive mental stress, 6 hours/day, 5 days per week for a total of 6 weeks.
  • the mouse atherosclerotic vulnerable plaque model was completed at 14 weeks postoperatively.
  • the experimental animals were randomly divided into the following groups, 6 in each group:
  • Vulnerable plaque model control group this group of animals did not undergo any therapeutic treatment
  • Rosuvastatin intragastric administration intragastric administration at a dose of 10 mg rosuvastatin / kg body weight;
  • Rosuvastatin intravenous group intravenous administration at a dose of 0.66 mg rosuvastatin / kg body weight;
  • PLGA-(R)-PEG-HA group intravenous administration at a dose of 0.66 mg rosuvastatin/kg body weight;
  • PLGA-(R)-PEG-SP group intravenous administration at a dose of 0.66 mg rosuvastatin/kg body weight;
  • PLGA-(R)-PEG-HA/Tat group intravenous administration was administered at a dose of 0.66 mg rosuvastatin/kg body weight.
  • the treatment group was treated once every other day for a total of 5 treatments.
  • carotid MRI scans were performed before and after treatment to detect plaque and lumen area, and the percentage of plaque progression was calculated.
  • Percentage of plaque progression (plaque area after treatment - plaque area before treatment) / lumen area.
  • Figure 21 shows the PLGA-(R)-PEG-HA, PLGA-(R)-PEG-SP, PLGA-(R)-PEG-HA/Tat vector delivery system of the present invention for arterial vulnerable plaque In vivo treatment effect.
  • the atherosclerosis of the control group (not given any treatment) progressed by 43%; rosuvastatin was used to treat the plaque.
  • PLGA-(R)-PEG-HA group eliminated plaque by 21%
  • PLGA-(R)-PEG-SP group eliminated plaque by 20%
  • PLGA-(R)-PEG-HA/ The Tat group eliminated plaque by 30%.
  • rosuvastatin has a certain therapeutic effect, whether it is administered by intragastric administration or intravenous administration, but it cannot prevent the damage.
  • the plaque continues to grow.
  • rosuvastatin was formulated in the nano-delivery system of the present invention, its therapeutic effect on vulnerable plaques was significantly improved, and treatment for reversing plaque growth (reducing plaque) was achieved. The effect is better with nano-systems with functional modifications.
  • Hyaluronic acid (HA) and IM7 are ligands for CD44, which can play a role in targeting vulnerable plaque.
  • Atorvastatin (At) has the effect of reversing plaque, and self-peptide (SEP) can increase drug penetration.
  • SEP self-peptide
  • miRNA-33a can increase cholesterol efflux.
  • the purpose of this example was to verify the PCL-(At)-PEG-HA, PCL-(At)-PEG-SEP/IM7, PCL-(At/miRNA-33a)-PEG-IM7 vector delivery system of the present invention. In vivo therapeutic effects on vulnerable plaque of arteries.
  • a physiological saline solution of free atorvastatin was formulated, and a micelle-loaded delivery system loaded with a therapeutic agent was prepared by the method described in the above Examples 4-6.
  • the experimental animals were randomly divided into the following groups, 6 in each group:
  • Vulnerable plaque model control group this group of animals did not undergo any therapeutic treatment
  • Atorvastatin gavage group intragastric administration at a dose of 20 mg atorvastatin / kg body weight;
  • Atorvastatin intravenous group intravenous administration of 1.2 mg atorvastatin / kg body weight;
  • PCL-(At)-PEG-HA group intravenous administration at a dose of 1.2 mg atorvastatin/kg body weight;
  • PCL-(At)-PEG-IM7 group intravenous administration at a dose of 1.2 mg atorvastatin/kg body weight;
  • PCL-(At)-PEG-SEP/IM7 group intravenous administration at a dose of 1.2 mg atorvastatin/kg body weight;
  • PCL-(At/miRNA-33a)-PEG-IM7 group intravenous administration treatment at a dose of 1.2 mg atorvastatin/kg body weight.
  • the treatment group was treated once every other day for a total of 5 treatments.
  • carotid MRI scans were performed before and after treatment to detect plaque and lumen area, and the percentage of plaque progression was calculated.
  • Percentage of plaque progression (plaque area after treatment - plaque area before treatment) / lumen area.
  • Figure 22 shows the PCL-(At)-PEG-HA, PCL-(At)-PEG-SEP/IM7, PCL-(At/miRNA-33a)-PEG-IM7 system of the present invention for arterial vulnerable plaques The in vivo therapeutic effect of the block.
  • the atherosclerosis of the control group progressed by 44.3%; the atorvastatin treatment can delay the plaque Progress, but also progressed 36.67%; atorvastatin intravenous injection also delayed plaque progression, but also progressed by 28.67%; while targeted nano drug-loading treatment significantly inhibited plaque progression, and even plaque Volume reversal and regression, PCL-(At)-PEG-HA group eliminated plaque 13.67%, PCL-(At)-PEG-IM7 group eliminated plaque 12.1%, PCL-(At)-PEG-SEP/ The IM7 group eliminated plaque by 18%, and the PCL-(At/miRNA-33a)-PEG-IM7 group eliminated plaque by 22%.
  • atorvastatin has a certain therapeutic effect, whether it is administered by intragastric administration or intravenous administration, but it cannot prevent the damage.
  • the plaque continues to grow.
  • atorvastatin was formulated in the nano-delivery system of the present invention, its therapeutic effect on vulnerable plaques was significantly improved, and treatment for reversing plaque growth (reducing plaque) was achieved. effect.
  • Nanocarriers modified with SEP function are more effective, while nanocarriers loaded with statins and nucleic acids are more effective.
  • Test Example 6 In vivo experiment of the effect of the PLGA-(AuNP/R)-PEG-OPN delivery system of the present invention on vulnerable plaque of arteries (CT tracing and treatment dual function)
  • Osteopontin is a ligand for CD44, which acts to target vulnerable plaques. Rosuvastatin (R) has the effect of reversing plaques. Nanogold (AuNP) is a CT tracer. The purpose of this example was to verify the in vivo tracing and therapeutic effects of the loaded CT tracer and rosuvastatin nano delivery system of the present invention on arterial vulnerable plaque.
  • micellar nano delivery system loaded with a CT tracer and a therapeutic agent was prepared by the method described in the above Example 7.
  • the experimental animals were randomly divided into the following groups, 6 in each group:
  • the group of free nano gold particles the dosage of nano gold is 0.1 mg/kg body weight;
  • PLGA-(AuNP/R)-PEG-OPN group nano gold was administered at a dose of 0.1 mg/kg body weight;
  • PLGA-(iodopramine)-PEG-OPN group iopromide is administered at a dose of 0.1 mg/kg body weight;
  • PLGA-(iodoxaxan)-PEG-OPN group iodixanol is administered at a dose of 0.1 mg/kg body weight
  • the PLGA-(iodofluoroalcohol)-PEG-OPN group iopromol was administered at a dose of 0.1 mg/kg body weight.
  • Each experimental group was injected with the corresponding tracer through the tail vein, and CT imaging was performed before administration and 2 hours after administration to observe the identification of atherosclerotic vulnerable plaque in each group.
  • the experimental animals were randomly divided into the following groups, 6 in each group:
  • Vulnerable plaque model control group this group of animals did not undergo any therapeutic treatment
  • Rosuvastatin intragastric administration intragastric administration at a dose of 10 mg rosuvastatin / kg body weight;
  • Rosuvastatin intravenous group intravenous administration at a dose of 0.67 mg rosuvastatin / kg body weight;
  • PLGA-(AuNP/R)-PEG-OPN group intravenous administration at a dose of 0.67 mg rosuvastatin/kg body weight;
  • the treatment group was treated once every other day for a total of 5 treatments.
  • carotid MRI scans were performed before and after treatment to detect plaque and lumen area, and the percentage of plaque progression was calculated.
  • Percentage of plaque progression (plaque area after treatment - plaque area before treatment) / lumen area.
  • Figure 23 illustrates the in vivo tracer effect of the load tracer-based micellar delivery system of the present invention on arterial vulnerable plaque.
  • the free nanogold particles showed a certain trace effect on arterial vulnerable plaques in mice.
  • nanogold, iopromide, iodixanol, and iodofluoroalcohol are formulated in a targeted micellar delivery system compared to free nanogold particles, their traceability to vulnerable plaques is Very significant improvement.
  • the use of the micelle delivery system with the surface-modified ligands described in the present invention can significantly improve the recognition of arterial vulnerable plaques by the use of the nanogold as compared to the free nanogold particles. Good tracer effect.
  • Figure 24 is a graph showing the in vivo therapeutic effect of the PLGA-(AuNP/R)-PEG-OPN system of the present invention on arterial vulnerable plaque.
  • the atherosclerosis of the control group (not given any treatment) progressed by 35%; rosuvastatin was used to treat the plaque.
  • the volume reversal and regression, PLGA-(AuNP/R)-PEG-OPN caused the plaque to fade by 18%.
  • rosuvastatin has a certain therapeutic effect, whether it is administered by intragastric administration or intravenous administration, but it cannot prevent the damage.
  • the plaque continues to grow.
  • rosuvastatin and nanogold are formulated in the nano-delivery system of the present invention, the diagnostic and therapeutic effects of vulnerable plaques are significantly improved, and early warning and reversal of high-risk patients are performed.
  • Test Example 7 In vivo tracer test (MRI tracing) and anti-inflammatory treatment of arterial vulnerable plaque by PLGA-(Fe 3 O 4 /DXMS)-PEG-HI44a delivery system of the present invention
  • Monoclonal antibody (HI44a) is an antibody against CD44 that targets vulnerable plaques.
  • Dexamethasone (DXMS) has anti-inflammatory effects and inhibits plaque progression.
  • Fe 3 O 4 is an MRI tracer. The purpose of this example was to verify the in vivo tracing and therapeutic effects of the loaded MRI tracer and dexamethasone nano-delivery system on arterial vulnerable plaques of the present invention.
  • gadolinium citrate, guanidine diamine, and guanidinic acid can also be prepared into nano-formulations, showing a targeted MRI tracer effect.
  • micellar nano delivery system loaded with an MRI tracer and a therapeutic agent was prepared by the method described in the above Example 8.
  • the experimental animals were randomly divided into the following groups, 6 in each group:
  • Free Fe 3 O 4 group Fe 3 O 4 is administered at a dose of 0.1 mg/kg body weight
  • PLGA-(Fe 3 O 4 /DXMS)-PEG-HI44a group Fe 3 O 4 is administered at a dose of 0.1 mg/kg body weight;
  • PLGA-(glucuronide)-PEG-HI44a group gadolinium citrate is administered at a dose of 0.1 mg/kg body weight;
  • PLGA-( ⁇ diamine)-PEG-HI44a group guanidine diamine is administered at a dose of 0.1 mg/kg body weight;
  • the PLGA-(capric acid)-PEG-HI44a group guanidonic acid was administered at a dose of 0.1 mg/kg body weight.
  • Each experimental group was injected with the corresponding tracer through the tail vein, and MRI imaging was performed before administration and 2 hours after administration to observe the identification of atherosclerotic vulnerable plaque in each group.
  • the experimental animals were randomly divided into the following groups, 6 in each group:
  • Vulnerable plaque model control group this group of animals did not undergo any therapeutic treatment
  • PLGA-(Fe 3 O 4 /DXMS)-PEG-HI44a group intravenous administration treatment at a dose of 0.1 mg dexamethasone/kg body weight;
  • the treatment group was treated once every other day for a total of 5 treatments.
  • carotid MRI scans were performed before and after treatment to detect plaque and lumen area, and the percentage of plaque progression was calculated.
  • Percentage of plaque progression (plaque area after treatment - plaque area before treatment) / lumen area.
  • Figure 25 illustrates the in vivo tracer effect of the load tracer-based micellar delivery system of the present invention on arterial vulnerable plaque.
  • the free Fe 3 O 4 particles exhibited a certain tracer effect on arterial vulnerable plaques in mice.
  • its traceability to vulnerable plaques is significantly improved, using other MRI nanometers. Contrast agents, the traceability of vulnerable plaques is also very good.
  • the use of the micelle delivery system with a surface-modified ligand described in the present invention significantly enhances the recognition of arterial vulnerable plaque by MRI tracers compared to free MRI tracers. , resulting in better tracer effects.
  • Figure 26 is a graph showing the in vivo therapeutic effect of the PLGA-(Fe 3 O 4 /DXMS)-PEG-HI44a system of the present invention on arterial vulnerable plaques.
  • the atherosclerosis of the control group (not given any treatment) progressed by 38%; while the targeted nano drug-loading treatment significantly suppressed the plaque.
  • the progression, and even the reversal and regression of plaque volume, PLGA-(Fe 3 O 4 /DXMS)-PEG-HI44a caused the plaque to fade by 5%.
  • Test Example 8 In vivo experiment of the effect of the PCL-(Asp/Clo)-PEG-Col delivery system of the present invention on vulnerable plaque of arteries
  • Aspirin (Asp) and clopidogrel (Clo) are antiplatelet agents that reduce platelet aggregation and reduce cardiovascular mortality.
  • the purpose of this example was to demonstrate the in vivo therapeutic effect of the PCL-(Asp/Clo)-PEG-Col vector delivery system of the present invention on arterial vulnerable plaque.
  • a physiological saline solution of free aspirin and clopidogrel was prepared, and a micelle-loaded delivery system loaded with a therapeutic agent was prepared by the method described in the above Example 9.
  • the experimental animals were randomly divided into the following groups, 10 in each group:
  • Plaque rupture model control group this group of animals did not undergo any therapeutic treatment
  • Aspirin and clopidogrel in the gavage group intragastric administration at a dose of 100 mg of aspirin/kg body weight and 75 mg of clopidogrel/kg body weight;
  • PCL-(Asp/Clo)-PEG-Col group intravenous administration treatment at a dose of 10 mg of aspirin/kg body weight and 7.5 mg of clopidogrel/kg body weight;
  • the treatment group was treated once every other day for a total of 5 treatments.
  • the mortality of the mice in January was observed, and the bleeding time (BT) of the mice was detected by tail-tailing.
  • Figure 27 is a graph showing the in vivo therapeutic effect of the PCL-(Asp/Clo)-PEG-Col system of the present invention on arterial vulnerable plaque.
  • mice in the control group had a mortality rate of 40%; intragastric administration with aspirin and clopidogrel reduced mortality to 20%; PCL-(Asp/Clo)- PEG-Col treatment can reduce mortality to 10%.
  • the PCL-(Asp/Clo)-PEG-Col group was not significantly prolonged, while the bleeding time was significantly prolonged in mice taking oral aspirin and clopidogrel.
  • oral dual antiplatelet therapy can reduce mortality, but prolong bleeding time and increase bleeding risk.
  • the loading of the antiplatelet drug into the nano delivery system of the present invention provides better efficacy than the oral drug without increasing the risk of bleeding.

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Abstract

一种用于靶向活化的CD44分子的胶束纳米载体递送系统，所述纳米载体的表面部分地被靶向配体修饰，所述靶向配体是能与活化的CD44分子特异性结合的配体。一种胶束纳米载体递送系统的制备方法和用途。该胶束纳米载体递送系统在制备用于诊断、预防和治疗易损斑块或与易损斑块相关疾病的药物中的用途。

Description

用于靶向活化CD44分子的胶束纳米载体递送系统、其制备方法和用途
相关申请的交叉引用
本申请要求2018年01月22日提交的第CN201810060265.9号中国发明专利申请的优先权，所述申请以引用的方式并入本文。
技术领域
本发明属于靶向给药技术领域，具体涉及一种用于靶向活化CD44分子，尤其是靶向易损斑块的纳米载体，尤其是胶束(Micelle)纳米递送系统。本发明也涉及所述纳米载体，尤其是胶束(Micelle)递送系统的制备方法和用途，特别是在易损斑块或与易损斑块相关的疾病的诊断、预防和治疗中的用途。
背景技术
目前，以急性心肌梗死和心源性猝死为主的急性心血管事件已成为危害人类健康的头号杀手。据统计，全世界每年约有2千万人死于急性心血管事件。在中国，情况同样不容乐观，每年有超过70万人死于急性心肌梗死和心源性猝死，这已经成为严重威胁我国居民健康的最重要疾病之一。研究表明，大部分的急性心肌梗死和心源性猝死均是由动脉粥样硬化斑块引起的。自上世纪70年代以来，人们一直在探索慢性动脉粥样硬化斑块导致急性冠脉综合征(ACS)及脑卒中的发生过程及机制。
1989年，Muller及其团队(Circadian variation and triggers of onset of acute cardiovascular disease.Circulation.1989；79(4):733-43)提出了“易损斑块”的概念，认为此类斑块是导致大多数急性心脑血管事件的根本原因。易损斑块(vulnerable plaque，又称为“不稳定斑块(unstable plaque)”)是指具有血栓形成倾向或极有可能快速进展成为“罪犯斑块”的动脉粥样硬化斑块，主要包括破裂斑块、侵蚀性斑块和部分钙化结节性病变。大量的研究表明，大部分的急性心肌梗死及脑卒中是由于轻、中度狭窄的易损斑块破裂，继发血栓形成所致。Naghavi及其团队(New developments in the detection of vulnerable plaque.Curr Atheroscler Rep.2001；3(2):125-35)等给出了易损斑块的组织学定义和标准。主要的标准包括活动性炎症、薄的纤维帽和大的脂质核心、内皮剥脱伴表面血小板聚集、斑块有裂隙或损伤以及严重的狭窄。次要的标准包括表面钙化斑、黄色有光泽的斑块、斑块内出血和正性重构。因此，对于易损斑块而言，早期干预至关重要。但是由于一般情况下易损斑块所导致的血管狭窄程度并不高，很多患者没有前驱症状，导致临床上很难进行早期诊断，使得其危险性极高。因此，如何尽早准确的识别和诊断易损斑块，进行有效的干预成为预防及治疗急性心肌梗死中亟待解决的问题。
目前常用于易损斑块诊断的技术主要包括冠脉造影、血管内超声(IVUS)、激光相干 断层显像(OCT)等技术，但这些技术均属于有创性的检查，并且诊断分辨率和准确性不高，同时这些诊断技术费用昂贵，也在一定程度上限制了临床上的普及。因此，目前急需针对易损斑块的无创诊断技术和制剂。
另外，目前治疗易损斑块的方法主要是全身给药，例如口服他汀类药物(羟甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂)、阿司匹林、基质金属蛋白酶(MMPs)抑制剂和/或贝特类药物等。这些药物通过调节全身血脂、对抗炎症、抑制蛋白酶和血小板生成等来减少斑块内的脂质，改善血管重构等，从而起到稳定斑块的作用。然而，临床应用中发现目前用于治疗易损斑块的药物的治疗效果并不理想。例如，临床常用的他汀类药物的口服给药生物利用度比较低，如辛伐他汀为＜5％，阿托伐他汀为约12％，瑞舒伐他汀为约20％。动物实验也证实，当他汀类药物的剂量增加到1mg/kg以上时才可以起到增加纤维帽厚度和减少斑块体积的作用，这就使得他汀类药物的口服给药的稳定性及逆转斑块的效果遭遇了瓶颈。目前临床试验也已经证实，口服他汀类药物治疗易损斑块需要采用强化大剂量才能具有稳定易损斑块的作用，而全身大剂量使用他汀类药物治疗也存在严重副作用(例如肝功能异常、横纹肌溶解、II型糖尿病等)发生率升高的风险。
对于现有的全身性给药而言，药物在进入体内后通常仅有极少一部分有效成分能够真正作用于病变部位。这是制约药物疗效，并导致药物毒副作用的根本原因。靶向给药系统是指具有靶向给药能力的给药系统。在经某种途径给药以后，靶向给药系统所包含的药物会通过带有靶向探针的载体特异性地富集于靶部位。靶向给药系统能够使药物瞄准特定的病变部位，并在目标病变部位释放有效成分。因此，靶向给药系统可以使药物在目标病变部位形成相对较高的浓度，并减少血液循环中的药量，从而在提高药效的同时抑制毒副作用，减少对正常组织和细胞的伤害。
目前，靶向给药系统通常所使用的纳米载体是脂质体。虽然脂质体具有提高药效、降低药物毒副作用的优势，但是由于其体内稳定性差，导致循环时间不足，最终对药物的生物利用度提升有限。另外，脂质体的体外稳定性同样不足，存储期间磷脂易氧化水解，而且脂质体囊泡之间容易相互聚集融合、包裹在其中的药物容易发生渗漏的问题。这均在一定程度上限制了靶向给药系统的发展。
另外，在易损斑块的诊断和治疗领域中，也存在一些利用靶向配体修饰纳米载体来诊断易损斑块的技术。然而，此类靶向易损斑块的靶向探针在临床实际应用中的主要问题在于这些制剂的靶向位点的特异性不足。例如，此类制剂的靶向位点大多选择巨噬细胞，但由于巨噬细胞可存在于身体各处，所以所述探针的靶向特异性不够理想。因此，靶向易损斑块的靶向制剂的研制中存在的难点在于发现易损斑块内的细胞中的具有显著靶向特异性的靶位。
CD44是一类黏附分子，广泛分布于淋巴细胞、单核细胞、内皮细胞等的表面。CD44分子的主要配体是透明质酸(hyaluronic acid，缩写为“HA”)。基于表达细胞的活化状态，可以将CD44分为相对静止状态(不能结合HA)、诱导活化状态(激活后可结合HA)和结构活跃状态(无需激活即可与HA结合)，而大多数正常细胞表面的CD44处于相对静止状态，从而不能与HA相结合。
继往大量研究表明CD44并不是具有显著靶向特异性的理想靶位。这是因为CD44在人体内广泛分布，尤其是大量存在于网状内皮丰富的器官表面上。因此，以CD44为靶位的靶向给药系统的研发中会遇到如下问题：如果靶位细胞表面的CD44与HA的亲和力不足以提供显著的特异性，那么此类靶向给药系统就不会存在特异性靶向性能。
因此，寻找易损斑块部位存在的特异性靶位以及适合于靶向易损斑块的靶向给药系统，由此开发能够特异性地靶向易损斑块，并同时能够实现药物的稳定持续释放的靶向给药系统，已经成为医学领域中的一个亟待解决的技术问题。
迄今为止，对于易损斑块内主要存在的巨噬细胞、单核细胞、内皮细胞、淋巴细胞和平滑肌细胞的表面上的CD44的表达状态及其与HA的亲和力尚无任何报道，也不存在任何关于利用HA和CD44的相互作用以及易损斑块的特定微环境设计用于诊断或治疗易损斑块或与易损斑块相关的疾病的能够实现药物的稳定持续释放的靶向给药系统的现有技术。
发明内容
(1)发明概述
发明人发现，与正常细胞相比，易损斑块中的细胞诸如内皮细胞、巨噬细胞和平滑肌细胞等的表面的CD44会被易损斑块的微环境(诸如在炎症因子的影响下)所诱导活化，导致与HA的结合能力会骤然增强数十倍。这一发现提示，易损斑块处的细胞表面存在的大量活化的CD44分子为以HA为靶向配体的靶向给药系统提供了理想的靶位。为此，本发明提供了一种能够特异性地靶向活化的CD44分子，尤其是靶向易损斑块的靶向给药系统。
发明人还发现，在易损斑块处存在大量的脂质如胆固醇等，这些脂质能够起到乳化剂的作用，会严重影响常规的靶向给药系统脂质体的稳定性，从而导致脂质体外层结构被快速地破坏或侵蚀(萃取)而崩解，这使得脂质体内包封的药物提前释放，无法起到药物持续释放的作用。然而，如果采用新型的药物递送系统纳米载体来代替脂质体，则会显著地改善包封药物在易损斑块处的释放特性，不会受到脂质侵蚀的影响，在易损斑块处的微环境中可以维持较好的稳定性，从而能够实现药物的持续释放。为此，本发明还提供一种能够特异性地靶向易损斑块，并同时能够实现药物的稳定持续释放的靶向给药系统。
发明人还发现，负载CD44活化剂可以促使病灶细胞表面的CD44进一步活化，可以在短时间内放大CD44对HA的靶向亲和力，显著增加结合在细胞表面的靶向组合物浓度，这对于易损斑块的示踪诊断和治疗具有积极意义。为此，本发明所述的靶向给药系统可以负载CD44活化剂，其可以在短时间内显著增加示踪剂或治疗剂化合物的浓度以提高诊断分辨率或治疗效果。
发明人还发现，在易损斑块内，伴随CD44的高度活化与过表达，同时内源性大分子HA也受激大量生成，并与细胞表面CD44结合，促进巨噬细胞和淋巴细胞等在易损斑块内的聚集。此种在细胞表面结合CD44的内源性HA会形成药物进入的屏障，会降 低药物的生物利用度。为此，本发明所述的靶向给药系统可以负载小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物，其通过与细胞表面的内源性HA的竞争性结合，解除内源性HA在细胞表面形成的屏障，有利于药物顺利进入病灶细胞内，显著提供治疗效果。
总而言之，本发明涉及如下方面：
本发明提供了一种用于靶向靶向活化的CD44分子的胶束纳米载体递送系统。
本发明提供了一种用于靶向易损斑块的胶束纳米载体递送系统。
本发明还提供了一种用于制备本发明所述的靶向易损斑块的纳米载体递送系统的方法。
本发明还提供了一种药物，其包含本发明所述的靶向易损斑块的纳米载体递送系统和药学上可接受的载体。
本发明还提供了一种诊断制剂，其包含本发明所述的靶向易损斑块的纳米载体递送系统。
本发明还提供了本发明所述的靶向易损斑块的纳米载体递送系统在制备用于预防和/或治疗易损斑块或与易损斑块相关的疾病的药物中的用途。
本发明还提供了本发明所述的靶向易损斑块的纳米载体递送系统在制备用于诊断易损斑块或与易损斑块相关的疾病的诊断制剂中的用途。
本发明还提供了一种用于预防和/或治疗易损斑块或与易损斑块相关的疾病的方法，所述方法包括向需要其的对象施用本发明所述的靶向易损斑块的纳米载体递送系统。
本发明还提供了一种用于诊断易损斑块或与易损斑块相关的疾病的方法，所述方法包括向需要其的对象施用本发明所述的靶向易损斑块的纳米载体递送系统。
本发明技术方案的具体实施方式及其含义将在下文中进行详细说明。
(2)技术术语及其含义
本文中所提及的术语具有以下含义：
“易损斑块”又称“不稳定斑块”，是指具有血栓形成倾向或极有可能快速进展成为“罪犯斑块”的动脉粥样硬化斑块，主要包括破裂斑块、侵蚀性斑块和部分钙化结节性病变。大量的研究表明，大部分的急性心肌梗死及脑卒中是由于轻、中度狭窄的易损斑块破裂，继发血栓形成所致。易损斑块的组织学表现包括活动性炎症、薄的纤维帽和大的脂质核心、内皮剥脱伴表面血小板聚集、斑块有裂隙或损伤以及严重的狭窄，以及表面钙化斑、黄色有光泽的斑块、斑块内出血和正性重构。
“与易损斑块相关的疾病”主要是指疾病的发生和发展过程中与“易损斑块”相关、以为“易损斑块”特征、由“易损斑块”引起或继发于“易损斑块”的疾病。“与易损斑块相关的疾病”主要包括动脉粥样硬化症、冠状动脉粥样硬化性心脏病(包括急性冠脉综合征、 无症状心肌缺血-隐匿性冠心病、心绞痛、心肌梗死、缺血性心脏病、猝死、支架内再狭窄)、脑动脉粥样硬化症(包括脑卒中)、外周血管动脉粥样硬化症(包括闭塞性周围动脉粥样硬化、视网膜动脉粥样硬化症、颈动脉粥样硬化症、肾动脉粥样硬化症、下肢动脉粥样硬化症、上肢动脉粥样硬化症、动脉粥样硬化性阳痿)、主动脉夹层、血管瘤、血栓栓塞、心力衰竭和心源性休克等疾病。
“靶向给药系统”是指具有靶向给药能力的给药系统。在经某种途径给药以后，靶向给药系统所包含的药物会通过特殊载体或靶向弹头(例如，靶向配体)的作用特异性地富集于靶部位。目前已知的用于实现靶向给药的手段包括利用各种微粒给药系统的被动靶向性能、在微粒给药系统的表面进行化学修饰、利用一些特殊的理化性能、利用抗体介导靶向给药、利用配体介导靶向给药、利用前体药物靶向给药等。其中，利用配体介导靶向给药是利用某些器官和组织上的特定的受体可与其特异性的配体发生专一性结合的特点，将药物载体与配体结合，从而将药物导向特定的靶组织。
“胶束纳米载体”是指，其中所述胶束的前体分子为同时具有亲水、疏水结构的嵌段高分子聚合物。亲水性片段可为人工合成高分子材料如聚乙二醇、聚氨基酸或亲水性天然高分子材料(透明质酸、壳聚糖、葡聚糖、普鲁兰多糖、海藻酸盐、肝素等)；疏水段通过聚合(接枝聚合、嵌段聚合)或化学键合的方式连接在亲水结构上，疏水结构的化学组成为聚苯醚(PPO)、聚碳酸酯(PC)、聚乳酸(PLA)、聚DL-丙交酯(PDLLA)、聚乳酸-羟基乙酸共聚物(PLGA)、聚己内酯(PCL)、聚天冬氨酸(pAsp)、焦谷氨酸(pGlu)等高分子结构，还可为小分子疏水结构如通过在上键合疏水结构域包括：5β-胆酸、1,5-双十四烷基-L-谷氨酸酯，双十六烷基胺、神经酰胺等疏水片段。这种两亲结构高分子材料便于在水相中自组装成胶束纳米载体，也便于在其表面偶联靶向配体。
“囊泡(Vesicles)”是某些两亲性分子分散于水中时会自发形成一类具有封闭双层结构的分子有序组合体，很多种类有类似结构的纳米载体，或者合成过程中的中间体都可以称为囊泡。
“透明质酸(hyaluronic acid，缩写为“HA”)”是一种高分子的聚合物，分子式：(C 14H 21NO 11)n。它是由单位D-葡萄糖醛酸及N-乙酰葡糖胺组成的高级多糖。D-葡萄糖醛酸及N-乙酰葡糖胺之间由β-1,3-配糖键相连，双糖单位之间由β-1,4-配糖键相连。透明质酸以其独特的分子结构和理化性质在机体内显示出多种重要的生理功能，如润滑关节，调节血管壁的通透性，调节蛋白质，水电解质扩散及运转，促进创伤愈合等。尤为重要的是，透明质酸具有特殊的保水作用，是目前发现的自然界中保湿性最好的物质。
“透明质酸的衍生物”在本文中是指任何能够保留透明质酸与易损斑块处的细胞表面上的CD44分子的特异性结合能力的透明质酸的衍生物，包括但不限于透明质酸的药学上可接受的盐、低级烷基(含有1-6个碳原子的烷基)酯、在体内能够经水解或其它方式形成透明质酸的前体药物等。判断某种物质是否是“透明质酸的衍生物”可以通过测定该物质与易损斑块处的细胞表面上的CD44分子的特异性结合能力来实现，这属于本领域技术人员的技能范围之内。
“CD44分子”是一类广泛地表达于淋巴细胞、单核细胞、内皮细胞等细胞的细胞膜 上的跨膜蛋白多糖黏附分子，由胞外区段、跨膜区段和胞内区段等三个区段构成。CD44分子可介导多种细胞与细胞、细胞与细胞外基质之间的相互作用，参与体内的多种信号的传导，从而改变细胞的生物学功能。CD44分子的主要配体是透明质酸，它与透明质酸之间的受体-配体结合决定了细胞在细胞外基质中的黏附和/或迁移。此外，CD44分子还参与透明质酸的代谢。
“约”代表在其后面给出的数值的±5％的范围内的所有值构成的集合。
(3)发明详述
本发明的第一方面提供了一种用于靶向活化的CD44分子的胶束纳米载体递送系统，所述纳米载体的表面部分地被靶向配体修饰，所述靶向配体是能与活化的CD44分子特异性结合的配体。
本发明的第二方面提供了一种用于靶向易损斑块的胶束纳米载体递送系统，所述纳米载体的表面部分地被靶向配体修饰，所述靶向配体是能与易损斑块处的细胞表面上的CD44分子特异性结合的配体。纳米载体表面还可以进行其他修饰，起到更好的效果。在载体表面修饰PEG，可以起到长循环的效果，延长药物的半衰期；在载体表面修饰穿膜肽，自身肽SEP，或者双重配体同时修饰，都可以起到放大药效的作用。
根据本发明第一方面或第二方面的胶束纳米载体递送系统，其中所述胶束纳米载体选自聚乳酸/聚乙二醇纳米(PLA-PEG)，聚己内酯/聚乙二醇载体(PEG-PCL)。
根据本发明第一方面或第二方面的纳米载体递送系统，其中，所述靶向配体选自GAG、胶原、层黏连蛋白、纤黏连蛋白、选择蛋白、骨桥蛋白(OPN)以及单克隆抗体HI44a，HI313，A3D8，H90，IM7，或透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物；
优选地，所述靶向配体选自胶原，透明质酸，选择蛋白，骨桥蛋白或单克隆抗体HI44a，IM7。
根据本发明第一方面或第二方面的纳米载体递送系统，其中，所述纳米载体负载有用于诊断、预防和/或治疗与出现CD44分子活化状况相关的疾病的物质。
根据本发明第一方面或第二方面的纳米载体递送系统，其中，所述纳米载体负载有用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的物质；
在一个实施方案中，所述物质是用于诊断易损斑块或与易损斑块相关的疾病的物质；
在一个实施方案中，所述用于诊断易损斑块或与易损斑块相关的疾病的物质是示踪剂；
在一个实施方案中，所述示踪剂选自CT示踪剂，MRI示踪剂和核素示踪剂；
在一个实施方案中，所述CT示踪剂选自碘纳米造影剂、金纳米造影剂、氧化钽纳米造影剂、铋纳米造影剂、镧系纳米造影剂，或其他类似结构的示踪剂；更优选为碘化造影剂或纳米金，或其他类似结构的示踪剂；进一步优选为碘海醇、碘卡酸、碘佛醇、碘克沙醇、碘普罗胺、碘比醇、碘美普尔、碘帕醇、碘昔兰、醋碘苯酸、胆影酸、碘苯扎酸、碘甘卡酸、泛影酸、碘他拉酸钠、碘苯酯、碘番酸、碘阿芬酸、醋碘苯酸钠、碘 多啥、丙碘酮、碘奥酮、碘曲仑、碘吡多、胆影酸葡甲胺、碘他拉酸、泛影葡胺、甲泛影酸、甲泛葡铵、碘化油或乙碘油，或其他类似结构的示踪剂，优选为纳米金；和/或
所述MRI示踪剂选自纵向弛豫造影剂和横向弛豫造影剂；更优选为顺磁性造影剂、铁磁性造影剂和超磁性造影剂；进一步优选为Gd-DTPA及其线型、环型多胺多羧类螯合物和锰的卟啉螯合物，大分子钆螯合物、生物大分子修饰的钆螯合物、叶酸修饰的钆螯合物、树状大分子显影剂、脂质体修饰的显影剂和含钆富勒烯，或其他类似结构的示踪剂；再优选为钆喷酸葡胺、钆特酸葡胺、钆贝葡胺、钆双胺、枸橼酸铁铵泡腾颗粒、顺磁性氧化铁(Fe 3O 4NPs)，或其他类似结构的示踪剂，优选为Fe 3O 4NPs；
所述核素示踪剂选自有碳14( 14C)、碳13( 13C)、磷32(32P)、硫35(35S)、碘131(131I)、氢3(3H)、锝99(99Tc)、氟18(18F)标记的氟代脱氧葡萄糖。
在一个实施方案中，所述物质是用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的药物、多肽、核酸和细胞因子中的一种或多种。
在一个实施方案中，所述物质是CD44活化剂；
在一个实施方案中，所述CD44活化剂是CD44抗体mAb或IL5、IL12、IL18、TNF-α、LPS。
在一个实施方案中，所述物质是小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物；
在一个实施方案中，所述小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物的分子量范围为1-500KDa，优选为1-20KDa，更优选为2-10KDa。
在一个实施方案中，所述纳米载体同时负载有用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的物质和CD44活化剂；
优选地，所述纳米载体同时负载有用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质和透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物；
更优选地，所述纳米载体同时负载有用于诊断易损斑块或与易损斑块相关的疾病的物质、用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质、任选的CD44活化剂和任选的分子量的透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物。
在一个实施方案中，所述物质是用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质；
优选地，所述用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质选自他汀类药物、贝特类药物、抗血小板药物、PCSK9抑制剂、抗凝药物、血管紧张素转换酶抑制剂(ACEI)、钙离子拮抗剂、MMPs抑制剂、β受体阻滞剂，糖皮质激素或其他的抗炎物质如IL-1抗体canakinumab，以及它们的药学上可接受的盐中的一种或多种，包括这些种类药物或物质的活性制剂，以及内源性的抗炎细胞因子比如白细胞介素10(IL-10))；
更优选地，所述用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质选自洛伐他汀、阿托伐他汀、瑞舒伐他汀、辛伐他汀、氟伐他汀、匹伐他汀、普伐他汀，苯扎贝特、环丙贝特、氯贝特、吉非贝齐、非诺贝特、普罗布考，抗PCSK9抗体如evolocumab、 alirocumab、bococizumab、RG7652、LY3015014和LGT-209，或adnectin如BMS-962476，反义RNAi寡核苷酸如ALN-PCSsc，核酸如microRNA-33a、microRNA-27a/b、microRNA-106b、microRNA-302、microRNA-758、microRNA-10b、microRNA-19b、microRNA-26、microRNA-93、microRNA-128-2、microRNA-144、microRNA-145反义链以及它们的核酸类似物如锁核酸，阿司匹林、阿西美辛、曲克芦丁、双嘧达莫、西洛他唑、盐酸噻氯匹定、奥扎格雷钠、氯吡格雷、普拉格雷、西洛他唑、贝列前素钠、替格瑞洛、坎格瑞洛、替罗非班、依替巴肽、阿昔单抗、普通肝素、克赛、速碧林、黄达肝葵钠、华法林、达比加群、利伐沙班、阿哌沙班、依度沙班、比伐卢定、依诺肝素、替他肝素、阿地肝素、双香豆素、硝酸香豆素、枸杞酸钠、水蛭素、阿加曲班，贝那普利、卡托普利、依那普利、培多普利、福辛普利、赖诺普利、莫昔普利、西拉普利、培哚普利、喹那普利、雷米普利、群多普利、坎地沙坦，依普罗沙坦、厄贝沙坦、氯沙坦、替米沙坦、缬沙坦、奥美沙坦、他索沙坦、硝苯地平、尼卡地平、尼群地平、氨氯地平、尼莫地平、尼索地平、尼伐地平、伊拉地平、非洛地平、拉西地平、地尔硫卓、维拉帕米、氯己定、米诺环素、MMI-166、美托洛尔、阿替洛尔、比索洛尔、普萘洛尔、卡维地络、巴马司他、马立马司他、普啉司他、BMS-279251、BAY 12-9566、TAA211、AAJ996A、nacetrapib、evacetrapib、Torcetrapib和Dalcetrapib，泼尼松、甲泼尼松、倍他米松、丙酸倍氯米松、得宝松、泼尼松龙、氢化可的松、地塞米松或其他的抗炎物质如IL-1抗体canakinumab)，以及它们的药效片段或药学上可接受的盐中的一种或多种，以及它们的药学上可接受的盐中的一种或多种，包括这些种类药物的活性结构片段，以及内源性的抗炎细胞因子比如白细胞介素10(IL-10)。
本发明的第三方面提供了一种用于制备第一方面或第二方面所述的用于靶向易损斑块的纳米递送系统的方法，所述方法包括以下步骤：
(1)将适量的胶束前体分子和用于诊断、预防和/或治疗易损斑块或与易损斑块相
关的疾病的物质溶解于合适的有机溶剂中，加入一定体积的水溶液，并进行探头超
声，得到载药胶束纳米递送系统；
(2)将载药胶束纳米颗粒在一定缓冲条件下与任选靶头分子进行化学键合。
(3)任选地通过透析法除去步骤(2)中得到的所述粗制悬浮液中所含有的未负载的用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的物质，得到负载的纳米递送系统。
本发明的第四方面提供了一种药物，所述药物包含第一方面或第二方面所述的胶束纳米载体递送系统以及药学上可接受的载体。
本发明的第五方面提供了一种诊断制剂，所述诊断制剂包含第一方面或第二方面所述的胶束纳米载体递送系统。
本发明的第六方面提供了第一方面或第二方面所述的胶束纳米载体递送系统、第四方面所述的药物、或第五方面所述的诊断制剂在制备用于预防和/或治疗与出现CD44分子活化状况相关的疾病的药物中的用途。
本发明的第七方面提供了第一方面或第二方面所述的纳米载体递送系统、第四方面 所述的药物、或第五方面所述的诊断制剂在制备用于预防和/或治疗与易损斑块或与易损斑块相关的疾病的药物和/或诊断制剂中的用途。
根据本发明第七方面的用途，所述易损斑块选自破裂斑块、侵蚀性斑块和部分钙化结节性病变中的一种或多种；
优选地，所述与易损斑块相关的疾病选自动脉粥样硬化症、冠状动脉粥样硬化性心脏病(包括急性冠脉综合征、无症状心肌缺血-隐匿性冠心病、心绞痛、心肌梗死、缺血性心脏病、猝死、支架内再狭窄)、脑动脉粥样硬化症(包括脑卒中)、外周血管动脉粥样硬化症(包括闭塞性周围动脉粥样硬化、视网膜动脉粥样硬化症、颈动脉粥样硬化症、肾动脉粥样硬化症、下肢动脉粥样硬化症、上肢动脉粥样硬化症、动脉粥样硬化性阳痿)、主动脉夹层、血管瘤、血栓栓塞、心力衰竭和心源性休克中的一种或多种。
本发明的第八方面提供了一种用于预防和/或治疗与出现CD44分子活化状况相关的疾病的方法，所述方法包括：对有需要的受试者给予第一方面或第二方面所述的纳米载体递送系统、第四方面所述的药物、或第五方面所述的诊断制剂。
本发明的第九方面提供了一种用于预防、诊断和/或治疗易损斑块或与易损斑块相关的疾病的方法，所述方法包括：对有需要的受试者给予第一方面或第二方面所述的纳米载体递送系统、第四方面所述的药物、或第五方面所述的诊断制剂。
优选地，所述易损斑块选自破裂斑块、侵蚀性斑块和部分钙化结节性病变中的一种或多种。
更优选地，所述与易损斑块相关的疾病选自动脉粥样硬化症、冠状动脉粥样硬化性心脏病(包括急性冠脉综合征、无症状心肌缺血-隐匿性冠心病、心绞痛、心肌梗死、缺血性心脏病、猝死、支架内再狭窄)、脑动脉粥样硬化症(包括脑卒中)、外周血管动脉粥样硬化症(包括闭塞性周围动脉粥样硬化、视网膜动脉粥样硬化症、颈动脉粥样硬化症、肾动脉粥样硬化症、下肢动脉粥样硬化症、上肢动脉粥样硬化症、动脉粥样硬化性阳痿)、主动脉夹层、血管瘤、血栓栓塞、心力衰竭和心源性休克中的一种或多种。
本发明的第十方面提供了一种用于诊断与出现CD44分子活化状况相关的疾病的方法，所述方法包括包括：对有需要的受试者给予第一方面或第二方面所述的纳米载体递送系统、第四方面所述的药物、或第五方面所述的诊断制剂。
总而言之，本发明所述的胶束纳米载体递送系统，对于出现CD44分子活化状况的疾病而言，具有如下优点：
1)本发明的胶束纳米载体递送系统能够特异性结合至活化的CD44分子，并能够实现药物的稳定持续释放。
2)易损斑块中的细胞表面CD44被细胞外基质微环境所诱导活化，大量过表达，并且CD44-HA的亲和力显著提高，使得易损斑块内的CD44与HA的相互作用具有极为显著的亲和特异性。由此，易损斑块内的CD44构成了本发明所述的靶向易损斑块的纳米载体递送系统的优良靶点。
3)本发明所述的靶向易损斑块的胶束纳米载体递送系统能够主动靶向进入易损斑 块内并与病灶细胞结合。因此，该递送系统可以实现所负载的物质在病灶处的持续释放，显著增加并持续保持病灶区域的物质浓度，从而提高该递送系统的诊断或治疗效果。
4)易损斑块内存在巨大的脂质池，其含有大量的氧化低密度脂蛋白(ox-LDL)。而脂质体在这样的内环境中是不稳定的，极易崩解从而不能实现可控释放的功能；而本发明所述的靶向易损斑块的纳米载体递送系统在易损斑块的脂质池中是相对稳定的，可以持续释放药物，从而保持病灶处的药物浓度。
5)本发明所述的靶向易损斑块的纳米载体递送系统还可以负载促CD44活化物质即CD44活化剂例如IL5、IL12、IL18、TNF-α、LPS。负载CD44活化剂可以促使病灶细胞表面的CD44进一步活化，可以在短时间内放大CD44对透明质酸的靶向亲和力，显著增加结合在细胞表面的靶向纳米载体组合物浓度，这对于易损斑块的示踪诊断和治疗具有积极意义，因为其可以在短时间内显著增加示踪剂或治疗剂化合物的浓度以提高诊断分辨率或治疗效果。
附图的简要说明
为了使本发明的内容得到更充分的理解，下面通过本发明的具体实施例并结合附图，对本发明作进一步详细地说明，其中：
图1为实施例1中PLGA-(R)-PEG-HA胶束纳米载体的扫描电镜(SEM)结果。
图2为实施例1中PLGA-(R)-PEG-HA胶束纳米载体的红外表征(FT-IR)结果。
图3为实施例2中PLGA-(R)-PEG-SP胶束纳米载体的红外表征(FT-IR)结果。
图4为实施例3中PLGA-(R)-PEG-HA/Tat胶束纳米载体的红外表征结果。
图5为实施例4中PCL-(At)-PEG-HA胶束纳米载体的红外表征结果。
图6为实施例5中PCL-(At)-PEG-SEP/IM7胶束纳米载体的红外表征结果。
图7为实施例6中PCL-(At)-Arg(miRNA-33a)-PEG-IM7透射电镜(TEM)图。
图8为实施例7中PLGA-(AuNP/R)-PEG-OPN透射电镜(TEM)图像。
图9为实施例7中PLGA-(AuNP/R)-PEG-OPN红外表征结果。
图10为实施例8中PLGA-(Fe 3O 4/DXMS)-PEG-HI44a的TEM结果。
图11为实施例8中PLGA-(Fe 3O 4/DXMS)-PEG-HI44a的红外表征结果。
图12为实施例9中PCL-(Asp/Clo)-PEG-Col的红外表征结果。
图13为试验例1中各类分子量的HA所制备的胶束纳米载体水合粒径随保存时间变化。
图14为试验例1中各类胶束纳米药物载体水合粒径随保存时间变化。
图15为试验例1中长期放置对药物包封率的影响。
图16为试验例1中各种胶束载体的体外释放药物结果，(A)前5小时(h)的药物释放率；(B)120小时内的药物释放率。
图17为试验例2中构建的小鼠动脉粥样硬化易损斑块模型的核磁共振成像图。
图18为模型小鼠的正常动脉血管壁内皮细胞和动脉易损斑块处内皮细胞表面的CD44含量测定结果(以半定量积分表示)图。
图19为模型小鼠的正常动脉血管壁内皮细胞和动脉易损斑块处内皮细胞表面的 CD44与HA的结合力测定结果(以结合力积分表示)图。
图20为模型小鼠的斑块外巨噬细胞和动脉易损斑块内巨噬细胞表面的CD44与HA的结合力测定结果(以结合力积分表示)图。
图21为本发明的PLGA-(R)-PEG-HA，PLGA-(R)-PEG-SP，PLGA-(R)-PEG-HA/Tat纳米递送系统对模型小鼠的颈动脉易损斑块的治疗效果图。
图22为PCL-(At)-PEG-HA，PCL-(At)-PEG-SEP/IM7，PCL-(At/miRNA-33a)-PEG-IM7纳米递送系统对模型小鼠的颈动脉易损斑块的治疗效果图。
图23为PLGA-(AuNP/R)-PEG-OPN及其他CT示踪剂纳米递送系统对模型小鼠的颈动脉易损斑块的体内示踪效果图。
图24为PLGA-(AuNP/R)-PEG-OPN纳米递送系统对模型小鼠的颈动脉易损斑块的治疗效果图。
图25为PLGA-(Fe3O4/DXMS)-PEG-HI44a及其他MRI示踪剂纳米递送系统对模型小鼠的颈动脉易损斑块的体内示踪效果图。
图26为PLGA-(Fe3O4/DXMS)-PEG-HI44a纳米递送系统对模型小鼠的颈动脉易损斑块的治疗效果图。
图27为PCL-(Asp/Clo)-PEG-Col纳米递送系统对模型小鼠动脉易损斑块破裂的治疗效果图。
为了进一步理解本发明，下面结合实施例对本发明的具体实施方案进行详细描述。但是应当理解，这些描述的目的只是为进一步说明本发明的特征和优点，而不构成对本发明的权利要求的任何限制。
具体实施方式
实施例1纳米载体PLGA-(R)-PEG-HA胶束递送系统的制备
本专利制备PLGA-PEG纳米载体包载药物瑞舒伐他汀(R)，利用胶束本身含有的透明质酸(HA)靶向分子实现靶向识别和药物递送。
1.1制备空白PLGA-PEG-NH2载体：
氨基化的PLGA-PEG(PLGA-PEG-NH2)购于西安瑞禧生物科技公司。取一定量PLGA-PEG-NH2材料溶于DMF中制成10mg mL-1溶液，将10m PLGA-PEG溶液与40mL纯水混合，并使用探头超声3min，获得均匀PLGA-PEG-NH2胶束纳米载体。
1.2负载瑞舒伐他汀(R)的PLGA-PEG-NH2纳米胶束(PLGA-(R)-PEG-NH2)的制备：
称取50mg PLGA-PEG-NH2和50mg瑞舒伐他汀共同溶解于50mL DMF中，将10m PLGA-PEG溶液与40mL纯水混合，并使用探头超声3min，获得负载瑞舒伐他汀的均匀PLGA-PEG-NH2胶束纳米载体(PLGA-(R)-PEG-NH2)。通过10K截留分子量的超滤管 纯化载体，除去为包载的瑞舒伐他汀游离药物，即可得到包载瑞舒伐他汀的载药纳米囊泡，冻干样品获得白色药物纯品。
1.3表面修饰HA的PLGA-(R)-PEG-NH2纳米载体(PLGA-(R)-PEG-HA)
称取分子量为1000D的HA粉末0.1g溶于10mL超纯水中，加入活化剂EDC 25mg，sulfo-NHS 20mg，室温活化反应30min。加入90mL无水乙醇沉淀活化好的HA分子(sulfo-NHS-HA)离心后除去上清，沉淀复溶于10mL水中，得到10mg mL-1的sulfo-NHS-HA溶液。
将制备的PLGA-(R)-PEG-NH2冻干粉溶于5mM的NaHCO3中，配成10mg mL-1溶液，取该溶液10mL加入1.0mL的10mg mL-1的sulfo-NHS-HA，室温搅拌反应24小时，通过100K截留分子量的超滤管纯化载体，除去未偶联的sulfo-NHS-HA，获得连接靶向分子HA并包载的瑞舒伐他汀的靶向载药纳米囊泡，冻干样品获得白色药物纯品(PLGA-(R)-PEG-HA)。
采用类似的方法，选用不同分子量的HA(1-500KDa)可以制备出多种胶束纳米载体。
图1为PLGA-(R)-PEG-HA胶束纳米载体的扫描电镜(SEM)结果。图2为PLGA-(R)-PEG-HA胶束纳米载体的红外表征(FT-IR)结果。
实施例2胶束靶向纳米载体递送系统PLGA-(R)-PEG-SP的制备
本专利制备PLGA-(R)-PEG-SP胶束靶向纳米载体包载药物瑞舒伐他汀(R)，利用纳米胶束载体本身含有的选择蛋白(SP)靶向分子实现靶向识别和药物递送。
2.1负载瑞舒伐他汀(R)的PLGA-PEG纳米胶束(PLGA-(R)-PEG)的制备：
氨基化的PLGA-PEG(PLGA-PEG-NH 2)购于西安瑞禧生物科技公司，称取50mg PLGA-PEG和50mg瑞舒伐他汀(R)共同溶解于10mL DMF中，将10mL PLGA-PEG溶液与40mL纯水混合，并使用探头超声3min获得负载瑞舒伐他汀的均匀胶束纳米载体(PLGA-(R)-PEG)。通过10K截留分子量的超滤管纯化载体，除去未包载的游离药物，即可得到包载R的载药纳米胶束，冻干样品获得白色药物纯品。
2.2表面修饰SP负载瑞舒伐他汀的PLGA-PEG纳米胶束(PLGA-(R)-PEG-SP)的制备
称取SP 1mg溶于1mL超纯水中，加入活化剂EDC 0.5mg，sulfo-NHS 0.5mg，室温活化反应30min。通过10K截留分子量的超滤管纯化活化的SP，除去小分子活化剂上清液复溶于1.0mL PBS缓冲溶液中，得到1mg mL -1的sulfo-NHS-SP溶液。
将制备的PLGA-(R)-PEG冻干粉溶于5mM的NaHCO 3中，配成10mg mL -1溶液，取该溶液10mL加入1.0mL的1mg mL -1的sulfo-NHS-SP，室温搅拌反应4小时，通过葡聚糖凝胶柱G-100纯化纳米药物，除去未偶联的sulfo-NHS-SP，获得连接靶向分子SP并包载的瑞舒伐他汀的靶向载药纳米胶束，冻干样品获得白色药物纯品(PLGA-(R)-PEG-SP)图3为PLGA-(R)-PEG-SP胶束纳米载体的红外表征(FT-IR)结果。
实施例3 PLGA-(R)-PEG-HA/Tat胶束靶向纳米载体递送系统的制备
本专利制备PLGA-(R)-PEG-HA/Tat纳米胶束包载药物瑞舒伐他汀(R)，纳米胶束本身含有的透明质酸HA和穿膜肽Tat，双重修饰在显著提升纳米载体的靶向富集能力，极大地提升靶向识别和药物递送效果。
3.1负载瑞舒伐他汀(R)的PLGA-PEG纳米胶束(PLGA-(R)-PEG-NH 2)的制备：
氨基化的PLGA-PEG(PLGA-PEG-NH 2)购于西安瑞禧生物科技公司，称取50mg PLGA-PEG和50mg瑞舒伐他汀(R)共同溶解于50mL DMF中，将10mg PLGA-PEG溶液与40mL纯水混合，并使用探头超声3min获得负载R的均匀胶束纳米载体(PLGA-(R)-PEG-NH2)。通过10K截留分子量的超滤管纯化载体，除去未包载的游离药物，即可得到包载R的胶束纳米载体，冻干样品获得白色药物纯品。
3.2表面双重修饰HA和Tat负载瑞舒伐他汀的PLGA-PEG纳米胶束(PLGA-(R)-PEG-HA/Tat)的制备
称取10mg HA(分子量1万)溶于1mL超纯水中，加入活化剂EDC 5.0mg，sulfo-NHS 5.0mg，室温活化反应30min。通过3K截留分子量的超滤管纯化活化，除去小分子活化剂沉淀复溶于10.0mL PBS缓冲溶液中，得到1mg mL-1的sulfo-NHS-HA溶液。
称取1.0mg Tat溶于1mL超纯水中，加入活化剂EDC 0.5mg，sulfo-NHS 0.5mg，室温活化反应30min。通过10K截留分子量的超滤管纯化活化的Tat，除去小分子活化剂沉淀复溶于1.0mL PBS缓冲溶液中，得到1mg mL-1的sulfo-NHS-Tat溶液。
将制备的PLGA-(R)-PEG冻干粉溶于5mM的NaHCO 3中，配成10mg mL-1溶液，取该溶液10mL加入0.5mL的1mg mL-1的sulfo-NHS-HA，以及0.5mL的1mg mL-1的sulfo-NHS-Tat室温搅拌反应4小时，通过G-100葡聚糖凝胶柱纯化纳米药物，除去未偶联的靶向分子，获得连接靶向分子并包载的瑞舒伐他汀的靶向载药纳米胶束，冻干样品获得白色药物纯品(PLGA-(R)-PEG-HA/Tat)。图4为PLGA-(R)-PEG-HA/Tat红外表征结果。
实施例4 PCL-(At)-PEG-HA胶束纳米载体递送系统的制备
本专利制备PCL-PEG胶束纳米载体包载药物阿托伐他汀(At)，利用胶束纳米载体本身含有的透明质酸(HA)靶向分子实现靶向识别和药物递送。
4.1制备空白PCL-PEG纳米胶束：
氨基化的PCL-PEG(PCL-PEG-NH2)购于西安瑞禧生物科技公司，取一定量PCL-PEG-NH2材料溶于DMF中制成10mg mL-1溶液，将10m PCL-PEG溶液与40mL纯水混合，并使用探头超声3min，获得均匀PCL-PEG胶束纳米载体。
4.2负载阿托伐他汀的PCL-PEG纳米胶束(PCL-(At)-PEG-NH2)的制备：
称取50mg PCL-PEG-NH2和50mg阿托伐他汀(At)共同溶解于50mL DMF中，将10m PCL-PEG-NH2溶液与40mL纯水混合，并使用探头超声3min，获得负载阿托伐他汀的均匀PCL-PEG-NH2胶束纳米载体(PCL-(At)-PEG-NH2)。通过10K截留分子量的超滤管纯化载体，除去未包载的阿托伐他汀游离药物，即可得到包载阿托伐他汀的载药纳米胶束，冻干样品获得白色药物纯品。
4.3表面修饰HA的PCL-(At)-PEG纳米载体(PCL-(At)-PEG-HA)
称取分子量为3万的HA粉末0.1g溶于10mL超纯水中，加入活化剂EDC 25mg，sulfo-NHS 20mg，室温活化反应30min。加入90mL无水乙醇沉淀活化好的HA分子(sulfo-NHS-HA)离心后除去上清，沉淀复溶于10mL水中，得到10mg mL-1的sulfo-NHS-HA溶液。
将制备的PCL-(At)-PEG-NH2冻干粉溶于5mM的NaHCO3中，配成10mg mL-1溶液，取该溶液10mL加入1.0mL的10mg mL-1的sulfo-NHS-HA，室温搅拌反应24小时，通过100K截留分子量的超滤管纯化载体，除去未偶联的sulfo-NHS-HA，获得连接靶向分子HA并包载的阿托伐他汀的靶向胶束纳米载体，冻干样品获得白色药物纯品(PCL-(At)-PEG-HA)。图5为PCL-(At)-PEG-HA红外表征结果。
实施例5 PCL-(At)-PEG-SEP/IM7胶束靶向纳米载体递送系统的制备
本专利制备PCL-(At)-PEG-SEP/IM7胶束纳米载体包载药物阿托伐他汀(At)，纳米胶束本身含有的自身肽SEP/IM7,双重修饰在显著提升纳米载体的靶向富集能力，极大地提升靶向识别和药物递送效果。
5.1负载阿托伐他汀(At)的PCL-PEG纳米胶束(PCL-(At)-PEG)的制备：
氨基化的PCL-PEG(PCL-PEG-NH2)购于西安瑞禧生物科技公司，称取50mg PCL-PEG和50mg阿托伐他汀(At)共同溶解于50mL DMF中，将10mg PCL-PEG溶液与40mL纯水混合，并使用探头超声3min获得负载At的均匀胶束纳米载体(PCL-(At)-PEG)。通过10K截留分子量的超滤管纯化载体，除去未包载的游离药物，即可得到包载At的载药纳米胶束，冻干样品获得白色药物纯品。
5.2表面双重修饰SEP/IM7负载阿托伐他汀的PCL-PEG纳米胶束(PCL-(At)-PEG-SEP/IM7)的制备
称取10mg自身肽(SEP)溶于1mL超纯水中，加入活化剂EDC 5.0mg，sulfo-NHS 5.0mg，室温活化反应30min。通过3K截留分子量的超滤管纯化活化，除去小分子活化剂沉淀复溶于1.0mL PBS缓冲溶液中，得到1mg mL-1的sulfo-NHS-SEP溶液。
称取1.0mg IM7溶于1mL超纯水中，加入活化剂EDC 0.5mg，sulfo-NHS0.5mg，室温活化反应30min。通过10K截留分子量的超滤管纯化活化的IM7，除去小分子活化剂沉淀复溶于1.0mL PBS缓冲溶液中，得到1mg mL-1的sulfo-NHS-IM7溶液。
将制备的PCL-(At)-PEG冻干粉溶于5mM的NaHCO3中，配成10mg mL-1溶液，取该溶液10mL加入1mL的1mg mL-1的sulfo-NHS-IM7，以及0.5mL的1mg mL-1的sulfo-NHS-SEP室温搅拌反应4小时，通过G-100葡聚糖凝胶柱纯化纳米药物，除去未偶联的sulfo-NHS-IM7和sulfo-NHS-SEP，获得连接靶向分子并包载的阿托伐他汀的靶向载药纳米胶束，冻干样品获得白色药物纯品(PCL-(At)-PEG-SEP/IM7)。图6为PCL-(At)-PEG-SEP/IM7红外表征结果。若无需修饰SEP，可将上述加入SEP的环节省去，得到未修饰SEP的PCL-(At)-PEG-IM7。
实施例6 PCL-(At/miRNA-33a)-PEG-IM7胶束靶向纳米载体递送系统的制备
本专利制备PCL-Arg-PEG纳米胶束包载药物阿托伐他汀微小RNA miRNA-33a，利用纳米胶束偶联的IM7靶向分子实现药物的靶向递送。
6.1空白PCL-Arg-PEG纳米胶束的制备
PCL-Arg-PEG-NH2购于西安瑞禧生物科技公司为PCL聚精氨酸及PEG的嵌段共聚物。取一定量PCL-Arg-PEG材料溶于DMF中制成10mg mL-1溶液，将10m PCL-Arg-PEG溶液与40mL纯水混合，并使用探头超声3min，获得均匀空白PCL-Arg-PEG胶束纳米载体。
6.3负载阿托伐他汀(At)的PCL-Arg-PEG纳米胶束(PCL-(At)-Arg-PEG)的制备：
称取50mg PCL-Arg-PEG-NH2和50mg阿托伐他汀(At)共同溶解于50mL DMF中，将10m PCL-Arg-PEG-NH2溶液与40mL纯水混合，并使用探头超声3min获得负载阿托伐他汀的均匀胶束纳米载体(PCL-(At)-PEG-NH2)。通过10K截留分子量的超滤管纯化载体，除去未包载的游离药物，即可得到包载阿托伐他汀的载药纳米胶束，冻干样品获得白色药物纯品。
6.4负载阿托伐他汀/微小核糖核酸(miRNA-33a)的纳米胶束(PCL-(At/miRNA)-PEG)的制备：
将制备的包载阿托伐他汀的载药纳米载体冻干粉溶于10mL 5mM的NaHCO3中配成10mg mL-1溶液，将miRNA-33a冻干粉溶解并加入纳米载体溶液中，室温反应2h。通过10K截留分子量的超滤管纯化载体，除去未结合的miRNA-33a，即可得到同时包载At/miRNA33-a的载药纳米胶束，冻干样品获得白色药物纯品。
6.5表面修饰IM7负载阿托伐他汀/反义微小核糖核酸(miRNA-33a)的纳米胶束(PCL-(At/miRNA-33a)-PEG-IM7)的制备
称取IM7 1mg溶于1mL超纯水中，加入活化剂EDC 1.0mg，sulfo-NHS 1.0mg，室温活化反应30min。通过10K截留分子量的超滤管纯化活化的IM7，除去小分子活化剂沉淀复溶于1.0mL PBS缓冲溶液中，得到1mg mL-1的sulfo-NHS-IM7溶液。
将制备的PCL-(At/miRNA-33)-PEG冻干粉溶于5mM的NaHCO3中，配成10mg mL-1溶液，取该溶液10mL加入1.0mL的1mg mL-1的sulfo-NHS-IM7，室温搅拌反应4小时，通过G-100葡聚糖凝胶柱纯化纳米药物，除去未偶联的sulfo-NHS-IM7，获得连接靶向分子IM7并包载的阿托伐他汀的靶向载药纳米胶束，冻干样品获得白色药物纯品(PCL-(At/miRNA-33a)-PEG-IM7)。图7为PCL-(At/miRNA-33a)-PEG-IM7的透射电镜(TEM)图。
实施例7 PLGA-(AuNP/R)-PEG-OPN胶束靶向纳米载体递送系统的制备
本专利制备PLGA-(AuNP/R)-PEG-OPN纳米胶束包载药物瑞舒伐他汀(R)和金纳米颗粒(AuNPs)，利用纳米胶束本身含有的骨桥蛋白(OPN)靶向分子实现靶向识别和药物递送，其中含有的AuNPs兼具CT成像功能。
7.1硫辛酸金纳米颗粒(TA-AuNPs)的制备
配置100mL 1mM的氯金酸水溶液，室温搅拌下加入1mL 0.1M的NaBH4即可得到金纳米颗粒。将溶液pH调至碱性，加入20mL 1mM的硫辛酸乙醇溶液，室温搅拌24小时，使用3K的超滤管纯化TA-AuNPs，即可得到疏水性的硫辛酸金纳米颗粒(TA-AuNPs)。
7.2负载瑞舒伐他汀(R)和金纳米颗粒(AuNPs)的PLGA-PEG纳米胶束 (PLGA-(AuNP/R)-PEG)的制备：
称取50mg PLGA-PEG-NH2,TA-AuNPs(0.1mg mL)和50mg瑞舒伐他汀共同溶解于50mL DMF中，将10mL PLGA-PEG溶液与40mL纯水混合，并使用探头超声3min，获得负载瑞舒伐他汀(R)和TA-AuNPs的均匀PLGA-PEG胶束纳米载体(PLGA-(AuNP/R)-PEG)。通过100K截留分子量的超滤管纯化载体，除去未包载的游离药物和TA-AuNPs，即可得到包载瑞舒伐他汀的载药纳米胶束，冻干样品获得药物纯品。
7.3表面修饰骨桥蛋白(OPN)的PLGA-(AuNP/R)-PEG-NH2纳米载体(PLGA-(AuNP/R)-PEG-OPN)
称取OPN 1.0mg溶于1mL超纯水中，加入活化剂EDC 1.0mg，sulfo-NHS 1.0mg，室温活化反应30min。通过10K截留分子量的超滤管纯化活化的OPN，除去小分子活化剂沉淀复溶于1.0mL PBS缓冲溶液中，得到1mg mL-1的sulfo-NHS-OPN溶液。
将制备的PLGA-(AuNP/R)-PEG冻干粉溶于5mM的NaHCO3中，配成10mg mL-1溶液，取该溶液10mL加入1.0mL的1mg mL-1的sulfo-NHS-OPN，室温搅拌反应4小时，通过G-100葡聚糖凝胶柱纯化纳米药物，除去未偶联的sulfo-NHS-OPN，获得连接靶向分子OPN并包载的瑞舒伐他汀的靶向载药纳米胶束，冻干样品获得白色药物纯品(PLGA-(AuNP/R)-PEG-OPN)。图8为PLGA-(AuNP/R)-PEG-OPN透射电镜(TEM)图像。图9为PLGA-(AuNP/R)-PEG-OPN红外表征结果。
实施例8 PLGA-(Fe 3O 4/DXMS)-PEG-HI44a胶束靶向纳米载体递送系统的制备
本专利制备PLGA-(Fe 3O 4/DXMS)-PEG-HI44a纳米胶束包载药物地塞米松(Dexamethasone，DXMS)，利用纳米胶束本身偶联的单抗(HI44a)靶向分子实现靶向识别和药物递送，负载Fe3O4 NPs实现MRI成像同时实现治疗作用。
8.1油酸包被的顺磁性氧化铁(Fe3O4 NPs)的制备
配置100mL 10mM三氯化铁(FeCl3)水溶液，剧烈搅拌条件下加入新制备0.1M氨水10mL，制得磁性铁纳米颗粒(Fe3O4 NPs)，将1mM的油酸乙醇溶液加入该反应体系中，搅拌24小时，获得通过油酸包被的顺磁性氧化铁(Fe3O4 NPs)。利用外加磁场纯化材料，真空干燥获得磁性材料粉末，重新分散于DMF中制得不同浓度的Fe3O4 NPs分散液。
8.2负载地塞米松(Dexamethasone，DXMS)和Fe3O4 NPs的PLGA-PEG纳米胶束(PLGA-(Fe3O4/DXMS)-PEG)的制备：
称取50mg PLGA-PEG-NH2,Fe3O4 NPs(0.1mg mL)和50mg地塞米松(DXMS)共同溶解于50mL DMF中，将10mL PLGA-PEG溶液与40mL纯水混合，并使用探头超声3min，获得负载DXMS和Fe3O4NPs的均匀PLGA-PEG胶束纳米载体(PLGA-(Fe3O4/DXMS)-PEG)。通过100K截留分子量的超滤管纯化载体，除去未包载的DXMS游离药物和Fe3O4 NPs，即可得到包载DXMS的载药纳米胶束，冻干样品获得药物纯品。
8.3表面修饰HI44a单克隆抗体的胶束纳米载体(PLGA-(Fe3O4/DXMS)-PEG-HI44a)
称取HI44a 1.0mg溶于1mL超纯水中，加入活化剂EDC 1.0mg，sulfo-NHS 1.0mg，室温活化反应30min。通过10K截留分子量的超滤管纯化活化的HI44a，除去小分子活化剂沉淀复溶于1.0mL PBS缓冲溶液中，得到1mg mL-1的sulfo-NHS-HI44a溶液。
将制备的PLGA-(Fe3O4/DXMS)-PEG冻干粉溶于5mM的NaHCO3中，配成10mg mL-1溶液，取该溶液10mL加入1.0mL的1mg mL-1的sulfo-NHS-HI44a，室温搅拌反应4小时，通过G-100葡聚糖凝胶柱纯化纳米药物，除去未偶联的sulfo-NHS-HI44a，获得连接靶向分子HI44a并包载Fe3O4NPs和DXMS和的靶向载药纳米胶束，冻干样品获得药物纯品。图10为PLGA-(Fe3O4/DXMS)-PEG-HI44a的TEM结果。图11为PLGA-(Fe3O4/DXMS)-PEG-HI44a的红外表征结果。
实施例9胶束靶向纳米载体递送系统PCL-(Asp/Clo)-PEG-Col的制备
本专利制备PCL-PEG胶束纳米载体包载药物阿司匹林(Asp)以及氯吡格雷(Clo)同时偶联靶向分子胶原(PCL-(Asp/Clo)-PEG-Col)，利用载体本身偶联的胶原靶向分子实现靶向识别和药物递送。
9.1负载阿司匹林/氯吡格雷的PCL-PEG纳米胶束(PCL-(Asp/Clo)-PEG)的制备：
称取50mg PCL-PEG-NH2和20mg阿司匹林(Asp)30mg氯吡格雷(Clo)共同溶解于10mL DMF中，将10m PCL-PEG-NH2溶液与40mL纯水混合，并使用探头超声3min，获得负载阿司匹林/氯吡格雷的均匀胶束纳米载体(PCL-(Asp/Clo)-PEG)。通过10K截留分子量的超滤管纯化载体，除去为包载游离药物，冻干样品获得白色药物纯品。
9.2表面修饰胶原蛋白(Col)的PCL-(Asp/Clo)-PEG纳米载体(PCL-(Asp/Clo)-PEG-Col)
称取胶原蛋白(Col)粉末10mg溶于1mL超纯水中，加入活化剂EDC 5mg，sulfo-NHS 5mg，室温活化反应30min。通过10K截留分子量的超滤管纯化活化的胶原，除去小分子活化剂，上清液复溶于10.0mL水中，得到1mg mL-1的sulfo-NHS-Col溶液。
将制备的PCL-(Asp/Clo)-PEG冻干粉溶于5mM的NaHCO3中，配成10mg mL-1溶液，取该溶液10mL加入1.0mL的10mg mL-1的sulfo-NHS-Col，室温搅拌反应24小时，通过100K截留分子量的超滤管纯化载体，除去未偶联的sulfo-NHS-Col，获得连接靶向分子Col并包载的药物的靶向纳米胶束，冻干样品获得白色药物纯品(PCL-(Asp/Clo)-PEG-Col)。图12为(PCL-(Asp/Clo)-PEG-Col的红外表征结果。
试验例1本发明的胶束纳米递送系统的性质考察
在本实施例中，以实施例1中制备的负载治疗剂的胶束纳米递送系统为例来证明本发明的递送系统具有稳定可控的性质，从而适合于易损斑块或与易损斑块相关的疾病的诊断、预防和治疗。
1.药物浓度测定法：
载体药物瑞舒伐他汀，阿托伐他汀、地塞米松、阿司匹林、氯吡格雷具有很强的紫外吸收特性，因此可以通过采用HPLC-UV法(使用Waters2487，沃特世公司(Waters Corporation)，美国)利用瑞舒伐他汀，阿托伐他汀、地塞米松、阿司匹林、氯吡格雷的 紫外吸收特性测定其含量。用不同浓度的瑞舒伐他汀，阿托伐他汀、地塞米松、阿司匹林、氯吡格雷溶液的浓度(X)对HPLC色谱峰的峰面积(Y)建立标准定量方程。
2.水合粒径的测定：
本发明的递送系统PLGA-(R)-PEG-HA,PLGA-(R)-PEG-SP,PLGA-(R)-PEG-HA/Tat，PCL-(At)-PEG-HA，PCL-(At)-PEG-SEP/IM7，PCL-(At/miRNA-33a)-PEG-IM7，PLGA-(AuNP/R)-PEG-OPN，PLGA-(Fe3O4/DXMS)-PEG-HI44a，PCL-(Asp/Clo)-PEG-Col的水合粒径均由激光粒度仪(BI-Zeta Plus/90 Plus，布鲁克海文公司(Brookhaven Instruments Corporation)，美国)测定，具体结果如表1所示。
3.包封率的测定：
用HPLC(Waters 2487，沃特世公司(Waters Corporation)，美国)测定所得液体中的药物含量，并通过公式1计算包封率。
取1.0mL悬浮液，通过加入过量甲醇破坏胶束结构，并进一步采用超声法，使药物加速从胶束中释放出来。用HPLC(Waters 2487，沃特世公司(Waters Corporation)，美国)测定所得液体中的药物含量，并通过公式1计算包封率，具体结果如表1所示。
Figure PCTCN2019072471-appb-000001
表1各种性质一览表
Figure PCTCN2019072471-appb-000002
Figure PCTCN2019072471-appb-000003
注：以上数据均以平行测定5次的结果的“平均值+标准差”的形式表示。
4.长期稳定性考察
将本发明的纳米递送系统PLGA-(R)-PEG-HA，PLGA-(R)-PEG-SP，PLGA-(R)-PEG-HA/Tat，PCL-(At)-PEG-HA，PCL-(At)-PEG-SEP/IM7，PCL-(At/miRNA-33a)-PEG-IM7，PLGA-(AuNP/R)-PEG-OPN，PLGA-(Fe 3O 4/DXMS)-PEG-HI44a，PCL-(Asp/Clo)-PEG-Col在4℃储存，于不同的时间点取样，并通过激光粒度仪(BI-Zeta Plus/90Plus，布鲁克海文公司(Brookhaven Instruments Corporation)，美国)检测其水合粒径的变化。
在放置90天后，PLGA-(R)-PEG-HA，PLGA-(R)-PEG-SP，PLGA-(R)-PEG-HA/Tat，PCL-(At)-PEG-HA，PCL-(At)-PEG-SEP/IM7，PCL-(At/miRNA-33a)-PEG-IM7，PLGA-(AuNP/R)-PEG-OPN，PLGA-(Fe 3O 4/DXMS)-PEG-HI44a，PCL-(Asp/Clo)-PEG-Col的平均水合粒径几乎保持不变，而且在整个试验期间未见分层、絮凝等现象。由此可见，这些载体均具有良好的储存稳定性，从而具有作为长循环靶向药物递送系统的应用潜力。图13为本专利设计的各类分子量的HA所制备的胶束纳米载体水合粒径随保存时间变化。图14为本专利设计的各类胶束纳米药物载体水合粒径随保存时间变化。
5.长期放置对包封率的影响将本发明的胶束递送系统放置60天后，PLGA-(R)-PEG-HA，PLGA-(R)-PEG-SP，PLGA-(R)-PEG-HA/Tat，PCL-(At)-PEG-HA，PCL-(At)-PEG-SEP/IM7，PCL-(At/miRNA-33a)-PEG-IM7，PLGA-(AuNP/R)-PEG-OPN，PLGA-(Fe 3O 4/DXMS)-PEG-HI44a，PCL-(Asp/Clo)-PEG-Col在在4℃储存，于不同时间点取样，通过超滤离心除去游离药物考察其包封率的变化。结果见图15。图15为长期放置对药物包封率的影响。
从图中可以看出，在放置60天后，胶束递送系统PLGA-(R)-PEG-HA，PLGA-(R)-PEG-SP，PLGA-(R)-PEG-HA/Tat，PCL-(At)-PEG-HA，PCL-(At)-PEG-SEP/IM7，PCL-(At/miRNA-33a)-PEG-IM7，PLGA-(AuNP/R)-PEG-OPN，PLGA-(Fe 3O 4/DXMS)-PEG-HI44a，PCL-(Asp/Clo)-PEG-Col药物包封率从起始72％到86％下降到46-53％。说明胶束结构能够较好的保护内部分子不被泄露。从以上数据可以充分地说明本发明的胶束递送系统的长期储存稳定性较好，在4℃储存两个月后粒径变化不大且药物的泄漏率较低。
6.体外释药性能研究
取2mL本发明的纳米递送系统PLGA-(R)-PEG-HA，PLGA-(R)-PEG-SP，PLGA-(R)-PEG-HA/Tat，PCL-(At)-PEG-HA，PCL-(At)-PEG-SEP/IM7，PCL-(At/miRNA-33a)-PEG-IM7，PLGA-(AuNP/R)-PEG-OPN，PLGA-(Fe 3O 4/DXMS)-PEG-HI44a，PCL-(Asp/Clo)-PEG-Col置于透析袋内密封。然后将 透析袋置于50mL释放介质(PBS溶液，pH＝7.4)中，于37℃孵育120h。在不同时间点取2mL释放液并补充相同体积的PBS溶液。用HPLC(Waters2487，沃特世公司(Waters Corporation)，美国)检测释放液中的药物含量，并通过公式3计算出药物的累积释放率。
Figure PCTCN2019072471-appb-000004
公式3中各参数意义如下：
CRP：药物累积释放率
V e：释放液的置换体积，此处V e为2mL
V 0：释放体系中释放液的体积，此处V 0为50mL
C i：第i次置换取样时释放液中药物的浓度，单位μg/mL
M 药物：递送系统中的药物的总质量，单位μg
n：置换释放液的次数
Cn：第n次置换释放液后测定的释放体系中的药物浓度。
体外释放是评价纳米粒子递送系统的一项重要指标。图16为本发明的胶束递送系统的药物累积释放率变化图。由图中可以看出，各类胶束递送系统在起始的5小时内释放速度较快，5小时药物释放率在19％到34％范围内。之后，药物释放速率逐渐变缓，经过60小时有60％到85％的药物被释放出来。前期较快的释药速率可能是由于部分吸附或沉淀胶束表面的可迅速溶解和扩散到释放介质中的药物的释放行为所导致的，而后期的药物释放主要是包封于胶束内的药物释放，表现为持续、缓慢的释放行为。体外释放实验结果显示药物从胶束中的释放能够被有效地延缓，体外释放实验结果说明胶束作为药物载体具有缓慢和持续的释放特性。
图16为各种胶束载体的体外释放药物结果，(A)前5小时(h)的药物释放率；(B)120小时内的药物释放率。
试验例2本发明的纳米递送系统的体内释放稳定性研究
在本实施例中，以实施例1中制备的负载瑞舒伐他汀的纳米递送系统为例来证明与脂质体递送系统相比，本发明的递送系统能够在易损斑块处保持相对稳定，从而实现长时间持续释放药物的效果。
实验方法：
取SPF级ApoE-/-小鼠(18只，10周龄，体重20±1g)作为实验动物。给予小鼠适应性高脂饮食(脂肪10％(w/w)，胆固醇2％(w/w)，胆酸钠0.5％(w/w)，其余部分为小鼠普通饲料)喂养4周后，用1％的戊巴比妥钠(配制方法为将1mg戊巴比妥钠加入至100ml 的生理盐水中)以40mg/kg的剂量腹腔注射麻醉。然后，将小鼠以仰卧位固定于手术板上，用75％(v/v)酒精以颈部为中心进行消毒，纵向剪开颈部皮肤，钝性分离颈前腺体，在气管的左侧可见搏动的左颈总动脉。小心分离颈总动脉至分叉处，将长度为2.5mm、内径为0.3mm的硅胶管套置于左颈总动脉的外周，套管的近心段和远心段均以细丝线缩窄固定。局部紧缩造成近端血流湍流，剪切力增加，造成血管内膜损伤。将颈动脉复位，间断缝合颈前皮肤。所有操作均在10倍体视显微镜下进行。术后待小鼠苏醒后将其放回笼中，维持环境温度在20～25℃，灯光保持开闭各12h。术后第4周开始腹腔注射脂多糖(LPS)(1mg/kg，在0.2ml磷酸盐缓冲盐水中，Sigma,USA)，每周2次，持续10周，诱导慢性炎症。术后8周将小鼠置入50ml注射器(预留充足通气孔)内造成限制性精神应激，6小时/天，每周5天，共持续6周。小鼠动脉粥样硬化易损斑块模型于术后14周造模完毕。图17中的(a)和(b)给出了所述小鼠动脉粥样硬化易损斑块模型的核磁共振成像图，由箭头指向部分可以看出左侧颈动脉斑块已经形成，提示造模成功，右侧劲动脉可作为正常动脉血管壁进行对比。
采用液相色谱-质谱法检测动脉易损斑块处的药物暴露百分比(其反映了注射实验药物后易损斑块处的瑞舒伐他汀的浓度随时间的变化)：
(1)标准溶液配制：
精密称取瑞舒伐他汀0.0141g，置于25mL容量瓶中，用甲醇溶解并稀释至刻度，摇匀，配制成浓度为56.4μg/mL的瑞舒伐他汀对照品储备液；将瑞舒伐他汀对照品储备液用甲醇稀释成10，1，0.5，0.125，0.05，0.025，0.01，0.002，0.0004μg/mL的系列标准溶液，4℃冷藏备用。
(2)内标溶液配制
精密称取对乙酰氨基酚0.0038g，置于25mL容量瓶中，用甲醇溶解并稀释至刻度，摇匀，配制成浓度为0.152mg/mL的对乙酰氨基酚储备液；将乙酰氨基酚储备液用甲醇稀释成15.2ng/mL的内标溶液，4℃冷藏备用。
(3)颈动脉样品前处理
分别于给药前和给药后2h，4h，8h，12h，24h，48h，72h，168h(七天)将动物处死(每个时间点一只小鼠)，迅速取出颈动脉斑块置于生理盐水中，用滤纸吸干表面水分，各剪取1cm，称量湿重，加1ml生理盐水匀浆，制成匀浆液。
取匀浆液1ml，加入甲醇20μL、浓度为15.2ng/mL的内标溶液100μL、10％(v/v)甲酸水溶液100μL、乙酸乙酯5mL，混匀，以14000rpm离心10min。取有机层溶液4ml，用氮气吹干。然后，用200μL流动相(0.1％(v/v)甲酸水溶液和乙腈(40:60，v/v))溶解，以14000rpm离心10min，取上清液，移入进样瓶，待测。
(4)标准曲线样品制备
取系列浓度的瑞舒伐他汀溶液10μL，加入500μL空白血浆，涡旋使其充分混匀，制备成浓度分别为200，20，10，2.5，1，0.5，0.2，0.04，0.008ng/mL的瑞舒伐他汀钙模拟含药血浆样品。按照血浆处理进行操作(加入15.2ng/mL内标溶液50μL，10％(v/v) 甲酸水溶液50μL，乙酸乙酯2.5mL，混匀，以14000rpm离心10min，取有机层溶液2ml，用氮气吹干，然后用100μL流动相溶解，以14000rpm离心10min，取上清液，移入进样瓶，待测)，建立标准曲线。以瑞舒伐他汀钙峰面积和内标峰面积比(y)为纵坐标，以血药浓度(x)为横坐标，用加权最小二乘法进行线性回归。
(5)液相色谱-质谱分析
液相分离采用Shimadzu modular LC system(东京，日本)系统进行，所述系统包括：1个DGU-20A3R真空脱气器，2个LC-20ADXR溶剂输送模块，1个SIL-20ACXR自动进样器，1个SPD-M20A PDA系统和1个CBM-20A控制器。该液相系统与装有ESI接口的ABSciex 5500 Qtrap质谱仪(Foster City,CA,USA)在线连接。Analyst软件(Version 1.6.2,ABSciex)用于数据采集与处理。
色谱分析采用CortecsTM UPLC C18柱(150mm×2.1mm内径(i.d.),1.6μm粒度)(Waters公司，美国)，柱温和样品室温度分别设为40℃和4℃。流动相为0.1％(v/v)甲酸水溶液和乙腈(40:60，v/v)，进样量为2μl。流速为0.2mL/min，单个样品分析时间为4min。
质谱检测采用的离子源为ESI源，正离子扫描模式。喷雾电压设为4500V，源温度设为500℃。通过多反应监测(MRM)方式检测各化合物，各成分离子通道分别为：瑞舒伐他汀m/z 482.2→258.2，对乙酰氨基酚m/z 152.2→110。优化各化合物碰撞能量和锥孔电压分别为：瑞舒伐他汀43V和100V，对乙酰氨基酚23V和100V。瑞舒伐他汀钙和对乙酰氨基酚的保留时间分别为2.07min和1.49min。
(6)标准曲线
瑞舒伐他汀的线性范围、相关系数(r)、线性方程及LLOQ如表2所示。从表中可以看出，瑞舒伐他汀钙的r值大于0.999，满足定量分析的要求。
表2瑞舒伐他汀钙的线性方程及LLOQ
Figure PCTCN2019072471-appb-000005
药物暴露百分比＝药物的重量/组织的重量
试验例3靶向机理的研究
在本实施例中，对易损斑块内皮细胞表面上的CD44的密度以及与HA之间的亲和力进行研究，从而为选择易损斑块内的CD44作为本发明所述的靶向易损斑块的递送系统的靶点提供了实验依据。
1)小鼠动脉易损斑块处内皮细胞表面与正常动脉血管壁内皮细胞表面的CD44含量比较：
按照上述实施例4中所述的方法，构建小鼠动脉粥样硬化易损斑块模型。取模型小鼠的正常动脉血管内皮细胞和动脉易损斑块处内皮细胞，采用免疫组织化学染色和图像分析方法进行CD44含量测定，具体实验方法如下：
取小鼠颈动脉粥样硬化易损斑块标本，经10mL/L甲醛水溶液固定、石蜡包埋、4μm切片、常规脱蜡、水化处理后，采用亲和素-生物素-酶复合物法(SABC)检测CD44含量。将标本浸入30mL/L H2O2水溶液以阻断内源性过氧化物酶的活性，并置入柠檬酸盐缓冲液中行抗原微波修复。然后滴加50g/L牛血清白蛋白(BSA)封闭液，于室温放置20min。然后，滴加鼠抗CD44多克隆抗体(1∶100)，于4℃冰箱放置过夜，再在37℃孵育1h。洗涤后，滴加生物素化的山羊抗鼠IgG，于37℃反应30min。然后用磷酸盐缓冲盐水(PBS)洗涤，并滴加辣根过氧化物酶标记的SABC复合物，于37℃孵育20min；以上每步均用PBS冲洗。最后用DAB显色(显色时在显微镜下控制)，随后用苏木素复染、脱水和封片。采用BI-2000图像分析系统的免疫组织化学分析系统分析切片，其中针对正常动脉血管内皮细胞和动脉易损斑块处内皮细胞组各采集3张切片，随机取5个具有代表性的视野。CD44表达阳性为：细胞膜、细胞质呈棕黄色/棕褐色且背景清晰，并且颜色越深说明CD44表达越强。未出现棕黄颗粒为CD44表达阴性。测量正常动脉血管内皮细胞和动脉易损斑块处内皮细胞组的阳性细胞平均吸光度(A)值并进行对比。结果如图18所示。
图18示出模型小鼠的正常动脉血管壁内皮细胞和动脉易损斑块处内皮细胞的表面CD44含量测定结果(以半定量积分表示)。如图所示，动脉易损斑块处内皮细胞的表面CD44含量约为正常动脉血管内皮细胞表面CD44含量的2.3倍。
2)小鼠动脉易损斑块处内皮细胞表面与正常动脉血管壁内皮细胞表面的CD44与配体及抗体的亲和力比较：
CD44的天然配体包括：HA、GAG、胶原、层黏连蛋白、纤黏连蛋白、选择蛋白、骨桥蛋白(OPN)以及单克隆抗体HI44a，HI313，A3D8，H90，IM7等。
取模型小鼠的正常动脉血管壁内皮细胞和动脉易损斑块处内皮细胞，加入浓度为10mg/ml的标记有氨基荧光素的配体/抗体，并用杜尔伯科改良伊格尔培养基(DMEM)培养基(含体积分数为10％的小牛血清、100U/ml青霉素、100U/ml链霉素)于37℃、5％CO 2培养箱中培养。30分钟后，利用流式细胞仪(CytoFLEX，贝克曼库尔特，美国)测定平均荧光强度(MFI)，并计算两种细胞表面的FL-配体/抗体的结合力积分(以正常动脉血管壁内皮细胞的CD44与配体/抗体的结合力为1)。结果如图19所示。
如图19所示，动脉易损斑块处内皮细胞表面的CD44与HA的结合力积分约为正常动脉血管壁内皮细胞表面的结合力积分的24倍。这表明正常动脉血管壁内皮细胞表面的CD44大多数处于不能与配体HA结合的静止状态，而动脉易损斑块处内皮细胞表面的CD44受到内环境中的诸如炎症因子等因素的影响而激活，与HA的亲和力显著增加。
CD44其他配体，与HA相似，易损斑块内皮细胞表面的CD44与GAG的结合力积分是正常细胞的22倍，易损斑块内皮细胞CD44与胶原的结合力积分是正常细胞的21倍，易损斑块内皮细胞CD44与层黏连蛋白的结合力积分是正常细胞的16倍，易损斑块内皮细胞CD44与纤黏连蛋白的结合力积分是正常细胞的18倍，易损斑块内皮细胞CD44 与选择蛋白的结合力积分是正常细胞的19倍，易损斑块内皮细胞CD44与骨桥蛋白的结合力积分是正常细胞的17倍。
CD44单克隆抗体也出现类似结果：易损斑块内皮细胞表面的CD44与H144a的结合力积分是正常细胞的15倍，易损斑块内皮细胞CD44与H1313的结合力积分是正常细胞的21倍，易损斑块内皮细胞CD44与A3D8的结合力积分是正常细胞的17倍，易损斑块内皮细胞CD44与H90的结合力积分是正常细胞的9倍，易损斑块内皮细胞CD44与IM7的结合力积分是正常细胞的8倍。
3)斑块外巨噬细胞和动脉易损斑块内巨噬细胞表面的CD44与配体/抗体的亲和力比较：
取模型小鼠的腹腔内的巨噬细胞和动脉易损斑块内的巨噬细胞，加入浓度为10mg/ml的标记有氨基荧光素的配体/抗体，用DMEM培养基(含体积分数为10％的小牛血清、100U/ml青霉素、100U/ml链霉素)于37℃、5％CO 2培养箱中培养。30分钟后，利用流式细胞计(CytoFLEX，贝克曼库尔特，美国)测定平均荧光强度(MFI)，并计算两种细胞表面上的FL-HA的结合力积分(以斑块外巨噬细胞表面的CD44与配体/抗体亲和力为1)。结果如图20所示。
如图20所示，动脉易损斑块内巨噬细胞表面的CD44-HA的结合力约为斑块外巨噬细胞表面的CD44-HA的结合力的40倍。这表明动脉易损斑块内的巨噬细胞表面的CD44同样受到内环境中的诸如炎症因子等因素的影响而激活，与HA的亲和力显著增加。
CD44其他配体，与HA相似，易损斑块巨噬细胞表面的CD44与GAG的结合力积分是正常细胞的33倍，易损斑块巨噬细胞CD44与胶原的结合力积分是正常细胞的38倍，易损斑块巨噬细胞CD44与层黏连蛋白的结合力积分是正常细胞的37倍，易损斑块巨噬细胞CD44与纤黏连蛋白的结合力积分是正常细胞的35倍，易损斑块巨噬细胞CD44与选择蛋白的结合力积分是正常细胞的33倍，易损斑块巨噬细胞CD44与骨桥蛋白的结合力积分是正常细胞的33倍。
CD44单克隆抗体也出现类似结果：易损斑块巨噬细胞表面的CD44与H144a的结合力积分是正常细胞的17倍，易损斑块巨噬细胞CD44与H1313的结合力积分是正常细胞的20倍，易损斑块巨噬细胞CD44与A3D8的结合力积分是正常细胞的16倍，易损斑块巨噬细胞CD44与H90的结合力积分是正常细胞的9倍，易损斑块巨噬细胞CD44与IM7的结合力积分是正常细胞的10倍。
综合上述实验的结果，可以得出如下结论：与正常细胞(诸如正常动脉血管壁内皮细胞、斑块外巨噬细胞)相比，易损斑块内的细胞(包括内皮细胞、巨噬细胞等，其对于动脉易损斑块的发展具有重要影响)表面上的CD44的密度显著提高，并且与配体的亲和力显著增强，从而导致动脉易损斑块内的CD44与配体的特异性亲和能力远远高于正常细胞，使其非常有利于作为本发明所述的靶向易损斑块的递送系统的优良靶点。
试验例4本发明的PLGA-(R)-PEG-HA，PLGA-(R)-PEG-SP，PLGA-(R)-PEG-HA/Tat瑞舒伐他汀递送系统对动脉易损斑块的影响的体内实验
透明质酸(HA)和选择蛋白(SP)是CD44的配体，能够起到靶向易损斑块的作用，瑞舒伐他汀(R)具有逆转斑块的作用，穿膜肽(Tat)能够增加药物局部穿透和聚集。本实施例的目的是验证本发明所述的PLGA-(R)-PEG-HA，PLGA-(R)-PEG-SP，PLGA-(R)-PEG-HA/Tat载体递送系统对动脉易损斑块的体内治疗作用。
实验方法：
(1)配制游离瑞舒伐他汀的生理盐水溶液，并采用上述实施例1-3中所述的方法制备负载治疗剂的胶束纳米递送系统。
(2)ApoE-/-小鼠动脉易损斑块模型的建立：
取SPF级ApoE-/-小鼠(42只，5-6周龄，体重20±1g)作为实验动物。给予小鼠适应性高脂饮食(脂肪10％(w/w)，胆固醇2％(w/w)，胆酸钠0.5％(w/w)，其余部分为小鼠普通饲料)喂养4周后，用1％的戊巴比妥钠(配制方法为将1mg戊巴比妥钠加入至100ml的生理盐水中)以40mg/kg的剂量腹腔注射麻醉。然后，将小鼠以仰卧位固定于手术板上，用75％(v/v)酒精以颈部为中心进行消毒，纵向剪开颈部皮肤，钝性分离颈前腺体，在气管的左侧可见搏动的左颈总动脉。小心分离颈总动脉至分叉处，将长度为2.5mm、内径为0.3mm的硅胶管套置于左颈总动脉的外周，套管的近心段和远心段均以细丝线缩窄固定。局部紧缩造成近端血流湍流，剪切力增加，造成的血管内膜损伤。将颈动脉复位，间断缝合颈前皮肤。所有操作均在10倍体视显微镜下进行。术后待小鼠苏醒后将其放回笼中，维持环境温度在20～25℃，灯光保持开闭各12h。术后第4周开始腹腔注射脂多糖(LPS)(1mg/kg，在0.2ml磷酸盐缓冲盐水中，Sigma,USA)，每周2次，持续10周，诱导慢性炎症。术后8周将小鼠置入50ml注射器(预留充足通气孔)内造成限制性精神应激，6小时/天，每周5天，共持续6周。小鼠动脉粥样硬化易损斑块模型于术后14周造模完毕。
(3)实验动物分组及治疗：
将实验动物随机分为以下各组，每组6只：
易损斑块模型对照组：该组动物不进行任何治疗性处理；
瑞舒伐他汀灌胃组：以10mg瑞舒伐他汀/kg体重的剂量进行灌胃给药处理；
瑞舒伐他汀静脉注射组：以0.66mg瑞舒伐他汀/kg体重的剂量进行静脉注射给药处理；
PLGA-(R)-PEG-HA组：以0.66mg瑞舒伐他汀/kg体重的剂量进行静脉注射给药处理；
PLGA-(R)-PEG-SP组：以0.66mg瑞舒伐他汀/kg体重的剂量进行静脉注射给药处理；
PLGA-(R)-PEG-HA/Tat组：以0.66mg瑞舒伐他汀/kg体重的剂量进行静脉注射给药处理。
除易损斑块模型对照组外，治疗组的治疗每隔一天进行1次，共治疗5次。对于各组动物，于治疗前后进行颈动脉MRI扫描以检测斑块和管腔面积，并计算斑块进展百分比。
斑块进展百分比＝(治疗后斑块面积-治疗前斑块面积)/管腔面积。
实验结果：
图21展示了本发明所述的PLGA-(R)-PEG-HA，PLGA-(R)-PEG-SP，PLGA-(R)-PEG-HA/Tat载体递送系统对动脉易损斑块的体内治疗效果。如图所示，高脂饮食喂养的过程中(10天)，对照组(不给于任何治疗处理)的动脉粥样硬化进展了43％；采用瑞舒伐他汀灌胃治疗，能够延缓斑块的进展，但也进展了34％；瑞舒伐他汀静脉注射同样延缓斑块进展，但也进展了23％；而靶向纳米载药治疗则明显遏制了斑块的进展，甚至出现了斑块体积的逆转和消退，PLGA-(R)-PEG-HA组使斑块消除21％，PLGA-(R)-PEG-SP组使斑块消除20％，PLGA-(R)-PEG-HA/Tat组使斑块消除30％。
综上所述，对于小鼠体内动脉易损斑块而言，无论是灌胃给药还是静脉注射给药，游离的瑞舒伐他汀都呈现出了一定的治疗效果，但是其无法阻止易损斑块的继续生长。然而，当将瑞舒伐他汀配制在本发明所述的纳米递送系统中时，其对于易损斑块的治疗效果发生了显著的提升，并起到了逆转斑块生长(缩小斑块)的治疗效果，带有功能修饰的纳米系统效果更佳。
试验例5本发明的PCL-(At)-PEG-HA，PCL-(At)-PEG-SEP/IM7，PCL-(At/miRNA-33a)-PEG-IM7递送系统对动脉易损斑块的影响的体内实验
透明质酸(HA)和IM7是CD44的配体，能够起到靶向易损斑块的作用，阿托伐他汀(At)具有逆转斑块的作用，自身肽(SEP)能够增加药物局部穿透和聚集，miRNA-33a能够增加胆固醇流出。本实施例的目的是验证本发明所述的PCL-(At)-PEG-HA，PCL-(At)-PEG-SEP/IM7，PCL-(At/miRNA-33a)-PEG-IM7载体递送系统对动脉易损斑块的体内治疗作用。
实验方法：
(1)配制游离阿托伐他汀的生理盐水溶液，并采用上述实施例4-6中所述的方法制备负载治疗剂的胶束纳米递送系统。
(2)ApoE-/-小鼠动脉易损斑块模型的建立方法同试验例4。
(3)实验动物分组及治疗：
将实验动物随机分为以下各组，每组6只：
易损斑块模型对照组：该组动物不进行任何治疗性处理；
阿托伐他汀灌胃组：以20mg阿托伐他汀/kg体重的剂量进行灌胃给药处理；
阿托伐他汀静脉注射组：以1.2mg阿托伐他汀/kg体重的剂量进行静脉注射给药处理；
PCL-(At)-PEG-HA组：以1.2mg阿托伐他汀/kg体重的剂量进行静脉注射给药处理；
PCL-(At)-PEG-IM7组：以1.2mg阿托伐他汀/kg体重的剂量进行静脉注射给药处理；
PCL-(At)-PEG-SEP/IM7组：以1.2mg阿托伐他汀/kg体重的剂量进行静脉注射给药处理；
PCL-(At/miRNA-33a)-PEG-IM7组：以1.2mg阿托伐他汀/kg体重的剂量进行静脉注射给药处理。
除易损斑块模型对照组外，治疗组的治疗每隔一天进行1次，共治疗5次。对于各组动物，于治疗前后进行颈动脉MRI扫描以检测斑块和管腔面积，并计算斑块进展百分比。
斑块进展百分比＝(治疗后斑块面积-治疗前斑块面积)/管腔面积。
实验结果：
图22展示了本发明所述的PCL-(At)-PEG-HA，PCL-(At)-PEG-SEP/IM7，PCL-(At/miRNA-33a)-PEG-IM7系统对动脉易损斑块的体内治疗效果。如图所示，高脂饮食喂养的过程中(10天)，对照组(不给于任何治疗处理)的动脉粥样硬化进展了44.3％；采用阿托伐他汀灌胃治疗，能够延缓斑块的进展，但也进展了36.67％；阿托伐他汀静脉注射同样延缓斑块进展，但也进展了28.67％；而靶向纳米载药治疗则明显遏制了斑块的进展，甚至出现了斑块体积的逆转和消退，PCL-(At)-PEG-HA组使斑块消除13.67％，PCL-(At)-PEG-IM7组使斑块消除12.1％，PCL-(At)-PEG-SEP/IM7组使斑块消除18％，PCL-(At/miRNA-33a)-PEG-IM7组使斑块消除22％。
综上所述，对于小鼠体内动脉易损斑块而言，无论是灌胃给药还是静脉注射给药，游离的阿托伐他汀都呈现出了一定的治疗效果，但是其无法阻止易损斑块的继续生长。然而，当将阿托伐他汀配制在本发明所述的纳米递送系统中时，其对于易损斑块的治疗效果发生了显著的提升，并起到了逆转斑块生长(缩小斑块)的治疗效果。采用SEP功能修饰的纳米载体效果更佳，同时负载他汀和核酸的纳米载体药效显著。
试验例6本发明的PLGA-(AuNP/R)-PEG-OPN递送系统对动脉易损斑块的影响的体内实验(CT示踪及治疗双功能)
骨桥蛋白(OPN)是CD44的配体，能够起到靶向易损斑块的作用，瑞舒伐他汀(R)具有逆转斑块的作用，纳米金(AuNP)是CT示踪剂。本实施例的目的是验证本发明所述的负载CT示踪剂和瑞舒伐他汀的纳米递送系统对动脉易损斑块的体内示踪及治疗效果。
(1)配制游离瑞舒伐他汀的生理盐水溶液，并采用上述实施例7中所述的方法制备负载CT示踪剂和治疗剂的胶束纳米递送系统。
(2)ApoE-/-小鼠动脉易损斑块模型的建立方法同试验例4。
(3)实验动物易损斑块示踪：
将实验动物随机分为以下各组，每组6只：
游离纳米金颗粒组：纳米金的给药剂量为0.1mg/kg体重；
PLGA-(AuNP/R)-PEG-OPN组：纳米金的给药剂量为0.1mg/kg体重；
PLGA-(碘普罗胺)-PEG-OPN组：碘普罗胺的给药剂量为0.1mg/kg体重；
PLGA-(碘克沙醇)-PEG-OPN组；碘克沙醇的给药剂量为0.1mg/kg体重；
PLGA-(碘氟醇)-PEG-OPN组：碘氟醇的给药剂量为0.1mg/kg体重。
各实验组分别经尾静脉注入相应的示踪剂，并于给药前以及给药后2h进行CT成像，观察各组动物的动脉粥样硬化易损斑块的识别情况。
(4)实验动物分组及治疗：
将实验动物随机分为以下各组，每组6只：
易损斑块模型对照组：该组动物不进行任何治疗性处理；
瑞舒伐他汀灌胃组：以10mg瑞舒伐他汀/kg体重的剂量进行灌胃给药处理；
瑞舒伐他汀静脉注射组：以0.67mg瑞舒伐他汀/kg体重的剂量进行静脉注射给药处理；
PLGA-(AuNP/R)-PEG-OPN组：以0.67mg瑞舒伐他汀/kg体重的剂量进行静脉注射给药处理；
除易损斑块模型对照组外，治疗组的治疗每隔一天进行1次，共治疗5次。对于各组动物，于治疗前后进行颈动脉MRI扫描以检测斑块和管腔面积，并计算斑块进展百分比。
斑块进展百分比＝(治疗后斑块面积-治疗前斑块面积)/管腔面积。
实验结果：
图23展示了本发明所述的负载示踪剂的胶束递送系统对动脉易损斑块的体内示踪效果。如图所示，游离的纳米金颗粒对于小鼠体内动脉易损斑块而言呈现出了一定的示踪效果。与游离的纳米金颗粒相比，当将纳米金，碘普罗胺，碘克沙醇，碘氟醇配制在靶向的胶束递送系统中时，其对于易损斑块的示踪效果有了非常显著的提高。综上所述，与游离纳米金颗粒相比，使用本发明所述的表面修饰有靶向配体的胶束递送系统给药可显著提高纳米金对动脉易损斑块的识别作用，产生更好的示踪效果。
图24展示了本发明所述的PLGA-(AuNP/R)-PEG-OPN系统对动脉易损斑块的体内治疗效果。如图所示，高脂饮食喂养的过程中(10天)，对照组(不给于任何治疗处理)的动脉粥样硬化进展了35％；采用瑞舒伐他汀灌胃治疗，能够延缓斑块的进展，但也进展了34％；瑞舒伐他汀静脉注射同样延缓斑块进展，但也进展了30％；而靶向纳米载药治疗则明显遏制了斑块的进展，甚至出现了斑块体积的逆转和消退，PLGA-(AuNP/R)-PEG-OPN使斑块消退了18％。
综上所述，对于小鼠体内动脉易损斑块而言，无论是灌胃给药还是静脉注射给药，游离的瑞舒伐他汀都呈现出了一定的治疗效果，但是其无法阻止易损斑块的继续生长。然而，当将瑞舒伐他汀和纳米金配制在本发明所述的纳米递送系统中时，其对于易损斑块的诊断和治疗效果发生了显著的提升，并起到了高危患者预警，以及逆转斑块生长(缩小斑块)的治疗效果。
试验例7本发明的PLGA-(Fe 3O 4/DXMS)-PEG-HI44a递送系统对动脉易损斑块的体内示踪实验(MRI示踪)及抗炎治疗
单克隆抗体(HI44a)是CD44的抗体，能够起到靶向易损斑块的作用，地塞米松(DXMS)具有抗炎，抑制斑块进展的作用，Fe 3O 4是MRI示踪剂。本实施例的目的是验证本发明所述的负载MRI示踪剂和地塞米松的纳米递送系统对动脉易损斑块的体内示踪及治疗效果。另外，钆特酸葡胺，钆双胺，钆喷酸也可以制备成纳米制剂，显示靶向MRI示踪效果。
(1)采用上述实施例8中所述的方法制备负载MRI示踪剂和治疗剂的胶束纳米递送系统。
(2)ApoE-/-小鼠动脉易损斑块模型的建立方法同试验例4。
(3)实验动物易损斑块示踪：
将实验动物随机分为以下各组，每组6只：
游离Fe 3O 4组：Fe 3O 4的给药剂量为0.1mg/kg体重
PLGA-(Fe 3O 4/DXMS)-PEG-HI44a组：Fe 3O 4的给药剂量为0.1mg/kg体重；
PLGA-(钆特酸葡胺)-PEG-HI44a组：钆特酸葡胺的给药剂量为0.1mg/kg体重；
PLGA-(钆双胺)-PEG-HI44a组：钆双胺的给药剂量为0.1mg/kg体重；
PLGA-(钆喷酸)-PEG-HI44a组：钆喷酸的给药剂量为0.1mg/kg体重。
各实验组分别经尾静脉注入相应的示踪剂，并于给药前以及给药后2h进行MRI成像，观察各组动物的动脉粥样硬化易损斑块的识别情况。
(4)实验动物分组及治疗：
将实验动物随机分为以下各组，每组6只：
易损斑块模型对照组：该组动物不进行任何治疗性处理；
PLGA-(Fe 3O 4/DXMS)-PEG-HI44a组：以0.1mg地塞米松/kg体重的剂量进行静脉注射给药处理；
除易损斑块模型对照组外，治疗组的治疗每隔一天进行1次，共治疗5次。对于各组动物，于治疗前后进行颈动脉MRI扫描以检测斑块和管腔面积，并计算斑块进展百分比。
斑块进展百分比＝(治疗后斑块面积-治疗前斑块面积)/管腔面积。
实验结果：
图25展示了本发明所述的负载示踪剂的胶束递送系统对动脉易损斑块的体内示踪效果。如图所示，游离的Fe 3O 4颗粒对于小鼠体内动脉易损斑块而言呈现出了一定的示踪效果。与游离的Fe 3O 4颗粒相比，当将Fe 3O 4配制在靶向的胶束递送系统中时，其对于易损斑块的示踪效果有了非常显著的提高，采用其他MRI纳米造影剂，易损斑块的示踪效果也很不错。综上所述，与游离MRI示踪剂相比，使用本发明所述的表面修饰有靶向配体的胶束递送系统给药可显著提高MRI示踪剂对动脉易损斑块的识别作用，产生更好的示踪效果。
图26展示了本发明所述的PLGA-(Fe 3O 4/DXMS)-PEG-HI44a系统对动脉易损斑块的 体内治疗效果。如图所示，高脂饮食喂养的过程中(10天)，对照组(不给于任何治疗处理)的动脉粥样硬化进展了38％；而靶向纳米载药治疗则明显遏制了斑块的进展，甚至出现了斑块体积的逆转和消退，PLGA-(Fe 3O 4/DXMS)-PEG-HI44a使斑块消退了5％。
综上所述，对于小鼠体内动脉易损斑块而言，当将地塞米松和Fe 3O 4配制在本发明所述的纳米递送系统中时，其对于易损斑块的诊断和治疗效果发生了显著的提升，并起到了高危患者预警，以及逆转斑块生长(缩小斑块)的治疗效果。
试验例8本发明的PCL-(Asp/Clo)-PEG-Col递送系统对动脉易损斑块的影响的体内实验
阿司匹林(Asp)和氯吡格雷(Clo)是抗血小板药物，能够起到减少血小板聚集的作用，可以减少心血管事件的死亡率。本实施例的目的是验证本发明所述的PCL-(Asp/Clo)-PEG-Col载体递送系统对动脉易损斑块的体内治疗作用。
实验方法：
(1)配制游离阿司匹林和氯吡格雷的生理盐水溶液，并采用上述实施例9中所述的方法制备负载治疗剂的胶束纳米递送系统。
(2)ApoE-/-小鼠动脉易损斑块破裂模型的建立：给予高脂饮食喂养30周，使ApoE-/-小鼠全身动脉形成动脉粥样硬化斑块，给予蛇毒诱导易损斑块破裂，形成急性冠脉综合征。
(3)实验动物分组及治疗：
将实验动物随机分为以下各组，每组10只：
斑块破裂模型对照组：该组动物不进行任何治疗性处理；
阿司匹林和氯吡格雷灌胃组：以100mg阿司匹林/kg体重和75mg氯吡格雷/kg体重的剂量进行灌胃给药处理；
PCL-(Asp/Clo)-PEG-Col组：以10mg阿司匹林/kg体重和7.5mg氯吡格雷/kg体重的剂量进行静脉注射给药处理；
除易损斑块模型对照组外，治疗组的治疗每隔一天进行1次，共治疗5次。对于各组动物，观察1月小鼠的死亡率，并断尾检测小鼠出血时间(BT)。
实验结果：
图27展示了本发明所述的PCL-(Asp/Clo)-PEG-Col系统对动脉易损斑块的体内治疗效果。如图所示，对照组(不给于任何治疗处理)的小鼠死亡率40％；采用阿司匹林和氯吡格雷灌胃治疗，能够使死亡率下降至20％；PCL-(Asp/Clo)-PEG-Col治疗能够使死亡率下降至10％。从出血时间来看，PCL-(Asp/Clo)-PEG-Col组未显著延长，而口服阿司匹林和氯吡格雷的小鼠出血时间显著延长。
综上所述，对于易损斑块破裂的动物而言，口服双抗血小板治疗可以降低死亡率，但是延长出血时间，增加出血风险。而本发明所述的将抗血小板药物负载至纳米递送系统，起到了比口服药物更好的疗效，且不增加出血风险。
已经通过上述实施例对本发明的各个方面进行了例示。显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (20)

  1. 一种用于靶向活化的CD44分子的胶束纳米载体递送系统，其特征在于，所述纳米载体的表面部分地被靶向配体修饰，所述靶向配体是能与活化的CD44分子特异性结合的配体；
    任选地，纳米载体表面可以进行其他修饰，所属修饰优选为在载体表面修饰PEG、穿膜肽、自身肽SEP中的一种或多种，或者双重配体同时修饰。
  2. 一种用于靶向易损斑块的胶束纳米载体递送系统，其特征在于，所述纳米载体的表面部分地被靶向配体修饰，所述靶向配体是能与易损斑块处的细胞表面上的CD44分子特异性结合的配体；
    任选地，纳米载体表面可以进行其他修饰，所属修饰优选为在载体表面修饰PEG、穿膜肽、自身肽SEP中的一种或多种，或者双重配体同时修饰。
  3. 根据权利要求1或2所述的纳米载体递送系统，其特征在于，所述胶束纳米载体选自聚乳酸\聚乙二醇纳米(PLA-PEG)和/或聚己内酯/聚乙二醇载体(PEG-PCL)。
  4. 根据权利要求1至3中任一项所述的纳米载体递送系统，其特征在于，所述靶向配体选自GAG、胶原、层黏连蛋白、纤黏连蛋白、选择蛋白、骨桥蛋白(OPN)以及单克隆抗体HI44a，HI313，A3D8，H90，IM7，或透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物；
    优选地，所述靶向配体选自胶原，透明质酸，选择蛋白，骨桥蛋白或单克隆抗体HI44a，IM7。
  5. 根据权利要求1至4中任一项所述的纳米载体递送系统，其特征在于，所述纳米载体负载有用于诊断、预防和/或治疗与出现CD44分子活化状况相关的疾病的物质。
  6. 根据权利要求1至5所述的纳米载体递送系统，其特征在于，所述纳米载体负载有用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的物质；
    优选地，所述所述物质是用于诊断易损斑块或与易损斑块相关的疾病的物质；
    更优选地，所述用于诊断易损斑块或与易损斑块相关的疾病的物质是示踪剂；
    进一步优选地，所述示踪剂选自CT示踪剂、MRI示踪剂和核素示踪剂；
    更进一步优选地：
    所述CT示踪剂选自碘纳米造影剂、金纳米造影剂、氧化钽纳米造影剂、铋纳米造影剂、镧系纳米造影剂，或其他类似结构的示踪剂；更优选为碘化造影剂或纳米金，或其他类似结构的示踪剂；进一步优选为碘海醇、碘卡酸、碘佛醇、碘克沙醇、碘普罗胺、碘比醇、碘美普尔、碘帕醇、碘昔兰、醋碘苯酸、胆影酸、碘苯扎酸、碘甘卡酸、泛影酸、碘他拉酸钠、碘苯酯、碘番酸、碘阿芬酸、醋碘苯酸钠、碘多啥、丙碘酮、碘奥酮、碘曲仑、碘吡多、胆影酸葡甲胺、碘他拉酸、泛影葡胺、甲泛影酸、甲泛葡铵、碘化油或乙碘油，或其他类似结构的示踪剂，优选为纳米金；
    所述MRI示踪剂选自纵向弛豫造影剂和横向弛豫造影剂；更优选为顺磁性造影剂、铁磁性造影剂和超磁性造影剂；进一步优选为Gd-DTPA及其线型、环型多胺多羧类螯合物和锰的卟啉螯合物，大分子钆螯合物、生物大分子修饰的钆螯合物、叶酸修饰的钆螯合物、树状大分子显影剂、脂质体修饰的显影剂和含钆富勒烯，或其他类似结构的示踪剂；再优选为钆喷酸葡胺、钆特酸葡胺、钆贝葡胺、钆双胺、枸橼酸铁铵泡腾颗粒、顺磁性氧化铁(Fe 3O 4NPs)，或其他类似结构的示踪剂，优选为Fe 3O 4NPS；和/或
    所述核素示踪剂选自有碳14( 14C)、碳13( 13C)、磷32(32P)、硫35(35S)、碘131(131I)、氢3(3H)、锝99(99Tc)、氟18(18F)标记的氟代脱氧葡萄糖。
  7. 根据权利要求6所述的纳米载体递送系统，其特征在于，所述物质是用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的药物、多肽、核酸和细胞因子中的一种或多种。
  8. 根据权利要求5至7中任一项所述的纳米载体递送系统，其特征在于，所述物质是CD44活化剂；
    优选地，所述CD44活化剂是CD44抗体mAb或IL5、IL12、IL18、TNF-α、LPS。
  9. 根据权利要求5至8中任一项所述的纳米载体递送系统，其特征在于，所述物质是小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物；
    优选地，所述小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物的分子量范围为1-500KDa，更优选为1-20KDa，最优选为2-10KDa。
  10. 根据权利要求6至9中任一项所述的纳米载体递送系统，其特征在于，所述纳米载体同时负载有用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的物质和CD44活化剂；
    优选地，所述纳米载体同时负载有用于预防和/或治疗易损斑块或与易损斑块相关的 疾病的物质和小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物；
    更优选地，所述纳米载体同时负载有用于诊断易损斑块或与易损斑块相关的疾病的物质、用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质、任选的CD44活化剂和任选的小分子透明质酸或能够与易损斑块处的细胞表面上的CD44分子特异性结合的透明质酸的衍生物。
  11. 根据权利要求6所述的纳米载体递送系统，其特征在于，所述物质是用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质；
    优选地，所述用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质选自他汀类药物、贝特类药物、抗血小板药物、PCSK9抑制剂、抗凝药物、血管紧张素转换酶抑制剂(ACEI)、钙离子拮抗剂、MMPs抑制剂、β受体阻滞剂，糖皮质激素或其他的抗炎物质如IL-1抗体canakinumab，以及它们的药学上可接受的盐中的一种或多种，包括这些种类药物或物质的制剂，以及内源性的抗炎细胞因子比如白细胞介素10(IL-10)；
    更优选地，所述用于预防和/或治疗易损斑块或与易损斑块相关的疾病的物质选自洛伐他汀、阿托伐他汀、瑞舒伐他汀、辛伐他汀、氟伐他汀、匹伐他汀、普伐他汀，苯扎贝特、环丙贝特、氯贝特、吉非贝齐、非诺贝特、普罗布考，抗PCSK9抗体如evolocumab、alirocumab、bococizumab、RG7652、LY3015014和LGT-209，或adnectin如BMS-962476，反义RNAi寡核苷酸如ALN-PCSsc，核酸如microRNA-33a、microRNA-27a/b、microRNA-106b、microRNA-302、microRNA-758、microRNA-10b、microRNA-19b、microRNA-26、microRNA-93、microRNA-128-2、microRNA-144、microRNA-145反义链以及它们的核酸类似物如锁核酸，阿司匹林、阿西美辛、曲克芦丁、双嘧达莫、西洛他唑、盐酸噻氯匹定、奥扎格雷钠、氯吡格雷、普拉格雷、西洛他唑、贝列前素钠、替格瑞洛、坎格瑞洛、替罗非班、依替巴肽、阿昔单抗、普通肝素、克赛、速碧林、黄达肝葵钠、华法林、达比加群、利伐沙班、阿哌沙班、依度沙班、比伐卢定、依诺肝素、替他肝素、阿地肝素、双香豆素、硝酸香豆素、枸杞酸钠、水蛭素、阿加曲班，贝那普利、卡托普利、依那普利、培多普利、福辛普利、赖诺普利、莫昔普利、西拉普利、培哚普利、喹那普利、雷米普利、群多普利、坎地沙坦，依普罗沙坦、厄贝沙坦、氯沙坦、替米沙坦、缬沙坦、奥美沙坦或他索沙坦、硝苯地平、尼卡地平、尼群地平、氨氯地平、尼莫地平、尼索地平、尼伐地平、伊拉地平、非洛地平、拉西地平、地尔硫卓、维拉帕米、氯己定、米诺环素、MMI-166、美托洛尔、阿替洛尔、比索洛尔、普萘洛尔、卡维地络、巴马司他、马立马司他、普啉司他、BMS-279251、BAY 12-9566、TAA211、AAJ996A、nacetrapib、evacetrapib、Torcetrapib和Dalcetrapib，泼尼松、甲泼尼松、倍他米松、丙酸倍氯米松、得宝松、泼尼松龙、氢化可的松、地塞米松，IL-1抗体canakinumab或其他的抗炎物质，以及它们的药效片段或药学上可接受的盐中的一种或多种，以及它们的药学上可接受的盐中的一种或多 种，包括这些种类药物的活性结构片段，以及内源性的抗炎细胞因子比如白细胞介素10(IL-10)；
    进一步优选为瑞舒伐他汀、阿托伐他汀、地塞米松、阿司匹林、氯吡格雷、microRNA-33a。
  12. 一种用于制备权利要求1至11中任一项所述的用于靶向易损斑块的纳米递送系统的方法，其特征在于，所述方法包括以下步骤：
    (1)将适量的胶束前体分子和用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的物质溶解于合适的有机溶剂中，加入一定体积的水溶液，并进行探头超声，得到载药胶束纳米递送系统；
    (2)将载药胶束纳米颗粒在一定缓冲条件下与任选靶头分子进行化学键合。
    (3)任选地通过透析法除去步骤(2)中得到的所述粗制悬浮液中所含有的未负载的用于诊断、预防和/或治疗易损斑块或与易损斑块相关的疾病的物质，得到负载的纳米递送系统。
  13. 一种药物，其特征在于，所述药物包含权利要求1至11中任一项所述的纳米载体递送系统以及药学上可接受的载体。
  14. 一种诊断制剂，其特征在于，所述诊断制剂包含权利要求1至11中任一项所述的纳米载体递送系统。
  15. 权利要求1至11中任一项所述的纳米载体递送系统、权利要求13所述的药物、或权利要求14所述的诊断制剂在制备用于预防和/或治疗与出现CD44分子活化状况相关的疾病的药物中的用途。
  16. 权利要求1至11中任一项所述的纳米载体递送系统、权利要求13所述的药物、或权利要求14所述的诊断制剂在制备用于预防和/或治疗与易损斑块或与易损斑块相关的疾病的药物和/或诊断制剂中的用途。
  17. 根据权利要求16所述的用途，其特征在于，所述易损斑块选自破裂斑块、侵蚀性斑块和部分钙化结节性病变中的一种或多种；
    优选地，所述与易损斑块相关的疾病选自动脉粥样硬化症、冠状动脉粥样硬化性心脏病(包括急性冠脉综合征、无症状心肌缺血-隐匿性冠心病、心绞痛、心肌梗死、缺血性心脏病、猝死、支架内再狭窄)、脑动脉粥样硬化症(包括脑卒中)、外周血管动脉粥样硬化症(包括颈动脉粥样硬化症、闭塞性周围动脉粥样硬化、视网膜动脉粥样硬化症、肾动脉粥样硬化症、下肢动脉粥样硬化症、上肢动脉粥样硬化症、动脉粥样硬化性阳痿)、主动脉夹层、血管瘤、血栓栓塞、心力衰竭和心源性休克中的一种或多种。
  18. 一种用于预防和/或治疗与出现CD44分子活化状况相关的疾病的方法，其特征在于，所述方法包括：对有需要的受试者给予权利要求1至11中任一项所述的纳米载体递送系统、权利要求13所述的药物、或权利要求14所述的诊断制剂。
  19. 一种用于预防、诊断和/或治疗易损斑块或与易损斑块相关的疾病的方法，其特征在于，所述方法包括：对有需要的受试者给予权利要求1至11中任一项所述的纳米载体递送系统、权利要求13所述的药物、或权利要求14所述的诊断制剂；
    优选地，所述易损斑块选自破裂斑块、侵蚀性斑块和部分钙化结节性病变中的一种或多种；
    更优选地，所述与易损斑块相关的疾病选自动脉粥样硬化症、冠状动脉粥样硬化性心脏病(包括急性冠脉综合征、无症状心肌缺血-隐匿性冠心病、心绞痛、心肌梗死、缺血性心脏病、猝死、支架内再狭窄)、脑动脉粥样硬化症(包括脑卒中)、外周血管动脉粥样硬化症(包括颈动脉粥样硬化症、闭塞性周围动脉粥样硬化、视网膜动脉粥样硬化症、肾动脉粥样硬化症、下肢动脉粥样硬化症、上肢动脉粥样硬化症、动脉粥样硬化性阳痿)、主动脉夹层、血管瘤、血栓栓塞、心力衰竭和心源性休克中的一种或多种。
  20. 一种用于诊断与出现CD44分子活化状况相关的疾病的方法，其特征在于，所述方法包括包括：对有需要的受试者给予权利要求1至11中任一项所述的纳米载体递送系统、权利要求13所述的药物、或权利要求14所述的诊断制剂。
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